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The development and evaluation of tourniquet-induced post-ischaemic allodynia : a method for investigating… Pau, Hin-Chung Clifford 2000

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THE D E V E L O P M E N T AND EVALUATION OF TOURNIQUET-INDUCED POST- ISCHAEMIC ALLODYNIA: A METHOD FOR INVESTIGATING THE MECHANISM OF AND T R E A T M E N T FOR CHRONIC PAIN by HIN-CHUNG CLIFFORD PAU B . S c , The University of British Columbia, 1998 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE D E G R E E OF MASTER OF S C I E N C E in THE FACULTY OF G R A D U A T E STUDIES (Department of Pharmacology & Therapeutics, Faculty of Medicine) We accept that this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA August 2000 © Hin-Chung Clifford Pau, 2000 In p resenting t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study. I f u r t h e r agree that permission f o r extensive copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the head of my department or by h i s or her r e p r e s e n t a t i v e s . I t i s understood that copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be allowed without my w r i t t e n permission. Department of -p [ A O X vAjU-g(L^ 0rV\ /7AX\ ^1 \\Q. The U n i v e r s i t y of B r i t i s h Columbia Vancouver, Canada Date A B S T R A C T Allodynia and hyperalgesia are two aspects of chronic pain for which understanding of their mechanisms and available treatments are currently inadequate. Currently available methods for investigating allodynia and hyperalgesia are generally invasive. The objective of this project was to evaluate tourniquet-induced post-ischaemic allodynia in mice - a potentially less invasive method for studying chronic pain. In the present study, a tourniquet was applied at the base of a mouse tail in order to induce post-ischaemic allodynia at the distal extremity. We quantified the tourniquet-induced post-ischaemic allodynia in mice with respect to its time course. The following two characteristics of post-ischaemic allodynia were observed with an increasing duration of tourniquet application: (1) Increase in onset time of the appearance of tourniquet-induced post-ischaemic allodynia, and (2) increase in the total duration of the tourniquet-induced post-ischaemic allodynia. The pharmacology associated with the induced allodynia was also investigated in this project. The effects of standard analgesic agents and local anaesthetic drugs were tested, and they were found to be effective in blocking pinprick pain and post-ischaemic allodynia in mice. However, none of the tested drugs were found to be effective in blocking the development of tourniquet-induced post-ischaemic allodynia in mice. We have also demonstrated how the method used to induce allodynia in mice could be applied to the study of the mechanism of and treatment for tourniquet-induced post-ischaemic allodynia. Systemic lidocaine was found to block both pinprick pain and post-ischaemic allodynia in mice centrally (in the spinal cord or higher level in the central nervous system) rather than peripherally (at the peripheral nerve endings), since its action was not prevented by exclusion of the drug from the site of pain generation. Morphine was equally effective in blocking pinprick pain and allodynic pain via an action presumptively only on the central nervous system, since its effect was not dependent on its reaching the site of pain generation. Morphine did not prevent the induction of post-ischaemic allodynia. TABLE OF CONTENTS A B S T R A C T ii T A B L E O F C O N T E N T S iv L IST O F F I G U R E S x L IST O F A B B R E V I A T I O N S xii 1. I N T R O D U C T I O N 1 1.1. Brief Introduction to Study ing Tourn iquet - Induced P o s t - i s c h a e m i c A l lodyn ia in M i c e 1 1.2. Def ini t ions 3 1.2.1. T a x o n o m y of Pa in 3 1.2.2. T a x o n o m y of A l lodyn ia and Hypera lges ia 5 1.3. B a c k g r o u n d 5 1.3.1. P a i n , A l lodyn ia and Hypera lges ia 5 1.3.2. Tourn iquet pain 7 1.3.3. M e t h o d s for eva luat ing a l lodynia and the von Frey hai rs 8 1.4. P resen t Unders tand ing about the M e c h a n i s m s of Pa in , A l lodyn ia and Hypera lges ia 10 1.4.1. P resen t Unders tand ing about Noc icept ion 10 1.4.1.1. Recep to r Leve l 10 1.4.1.2. C h e m i c a l Med ia to rs 10 1.4.1.3. Neurona l Leve l 11 1.4.1.4. C N S Leve l 12 1.4.1.5. Psycho log i ca l Fac to rs 14 1.4.2. P resen t unders tanding of a l lodynia and hypera lges ia 15 1.4.2.1. Per iphera l m e c h a n i s m s 16 1.4.2.2. Cent ra l m e c h a n i s m s 17 1.4.3. Current ly used s tandard ana lges i c agents and their m e c h a n i s m s of ac t ions 18 1.4.3.1. Op io id A n a l g e s i c s 18 iv 1.4.3.2. Loca l Anaes the t i c s 19 1.4.3.3. G e n e r a l A n a e s t h e t i c s / G e n e r a l D e p r e s s a n t s 21 1.4.3.4. N e u r o m u s c u l a r Junct ion B locke r 21 1.4.3.5. Non-s tero ida l Ant i - in f lammatory Drugs ( N S A I D s ) 22 M A T E R I A L S A N D M E T H O D S 24 2.1. Exper imen ta l Defini t ion of P a i n and A l lodyn ia 2 4 2.2. Mater ia ls 24 2.2 .1 . Exper imen ta l Sub jec ts 24 2.2.2. T h e Dev i ce for Inducing A l lodyn ia in a M o u s e Tai l : T h e Tourn iquet 26 2.2.3. T h e Appa ra tus for Inducing Pa in in M ice : P inpr ick Dev i ce 26 2.2.4. T h e Appa ra tus for A s s e s s i n g A l lodyn ia in M ice : T h e V o n Frey F i laments 27 2.2.5. T h e Dev i ce for A s s e s s i n g Motor Funct ion of M ice : T h e Rota rod 28 2.2.6. S tandard A n a l g e s i c Agen t s T e s t e d 33 2.3. Me thods 33 2.3 .1 . P h a s e I: Investigation of Tourn iquet Induced P o s t - i s c h a e m i c A l lodyn ia 35 2.3 .1 .1 . T h e Re la t ionsh ip be tween the Durat ion of Tourn iquet App l ica t ion and the Induced P o s t - i s c h a e m i c A l lodyn ia 35 2.3.1.2. Ef fects of I schaemia on Pinpr ick Pa in 36 2.3.2. P h a s e II: Ef fects of S tandard A n a l g e s i c A g e n t s on Pinpr ick Pa in in No rma l M i c e 36 2.3 .2 .1 . T h e Ef fects of S y s t e m i c Adminis t rat ion of the A n a l g e s i c Drugs on Pinpr ick Pa in in Norma l M i c e 36 2.3.2.2. T h e Ef fects of S y s t e m i c Admin is t ra t ion (with the Tai l O c c l u d e d by a Tourniquet) of the A n a l g e s i c Drugs on Pinpr ick Pa in in Norma l M i c e 37 2.3 .2 .3 . T h e Ef fec ts of Per iphera l Admin is t ra t ion of the A n a l g e s i c D rugs o n P inpr ick P a i n in Norma l M i c e 38 2.3.3. P h a s e III: Ef fects of S tandard A n a l g e s i c Agen t s on Tourn iquet - Induced P o s t - i s c h a e m i c A l lodyn ia in M i c e 39 2.3 .3 .1 . T h e Ef fects of S y s t e m i c Adminis t rat ion of A n a l g e s i c A g e n t s on Tourn iquet - Induced P o s t - i s c h a e m i c A l lodyn ia in M i c e 39 2.3.3.2. T h e Ef fects of S y s t e m i c Admin is t ra t ion (with the Tai l O c c l u d e d by a Tourniquet) of A n a l g e s i c A g e n t s on Tourn iquet - Induced P o s t - i s c h a e m i c A l lodyn ia in M i c e 40 v 2.3.3.3. T h e Ef fects of Per iphera l Adminis t rat ion of A n a l g e s i c A g e n t s on Tourn iquet - Induced P o s t - l s c h a e m i c A l lodyn ia in M i c e 41 2.3.4. P h a s e IV: E f fec ts of S tanda rd A n a l g e s i c A g e n t s o n the D e v e l o p m e n t of Tourn iquet -Induced P o s t - l s c h a e m i c A l lodyn ia in M i c e 42 2 .3 .4 .1 . T h e Ef fects of S y s t e m i c Admin is t ra t ion of A n a l g e s i c Drugs on the Deve lopmen t of Tourn iquet - Induced P o s t - l s c h a e m i c A l lodyn ia in M i c e 42 2.3.4.2. T h e Ef fects of Inhalation G e n e r a l Anaes the t i cs on the Deve lopmen t of Tourn iquet -Induced P o s t - l s c h a e m i c A l lodyn ia in M i c e 43 2.3.4.3. T h e Ef fects of Per iphera l Admin is t ra t ion of A n a l g e s i c Drugs on the Deve lopmen t of P o s t - l s c h a e m i c A l lodyn ia in M i c e 4 3 2.3.5. Da ta Ana l ys i s for P h a s e II, III and IV of the Study 4 5 R E S U L T S 46 3.1. P h a s e I: Investigation of Tourn iquet - Induced P o s t - l s c h a e m i c A l lodyn ia in M i c e 46 3.1.1. T i m e C o u r s e of P o s t - l s c h a e m i c A l lodyn ia 46 3.1.1 .1 . La tency of the A p p e a r a n c e of P o s t - l s c h a e m i c A l lodyn ia 47 3.1.1.2. Durat ion of P o s t - l s c h a e m i c A l lodyn ia 49 3.1.1.3. R e c o v e r y f rom Tourn iquet - Induced P o s t - l s c h a e m i c A l lodyn ia 59 3.1.2. Ef fects of I schaemia on Pinpr ick Pa in 66 3.2. P h a s e II: Ef fects of S tandard A n a l g e s i c A g e n t s on Pinpr ick Pa in 69 3.2.1. T h e Ef fec ts of S y s t e m i c Admin is t ra t ion of A n a l g e s i c Drugs on Pinpr ick Pa in in Norma l M i c e 69 3.2.1.1. Morph ine (5 mg/kg , 10 mg/kg , and 20 mg/kg) 70 3.2.1.2. L idoca ine (40 mg/kg , 75 mg/kg) , Q X - 2 2 2 (140 mg/kg) , and Bup ivaca ine (50 mg/kg) 72 3.2.1.3. Pentobarb i ta l (30 mg/kg , 40 mg/kg) 79 3.2.1.4. R o c u r o n i u m (0.7 mg/kg) 81 3.2.2. T h e Ef fects of S y s t e m i c Admin is t ra t ion (with the Tai l O c c l u d e d by a Tourniquet) of A n a l g e s i c Drugs on Pinpr ick Pa in in Norma l M i c e 82 3.2.2.1. L idoca ine (75 mg/kg) 82 3.2.3. T h e Ef fec ts of Per iphera l Admin is t ra t ion of A n a l g e s i c D r u g s on P inpr ick P a i n in N o r m a l M i c e 85 3.2.3 .1 . Morph ine (1.5%) 85 vi 3.2.3.2. L idoca ine (1%), Bup ivaca ine (0.3%), and Q X - 3 1 4 (2%) 86 3.2.3.3. A S A (2.4%) 87 3.3. P h a s e III: Ef fects of S tandard A n a l g e s i c A g e n t s on Tourn iquet - Induced P o s t - i s c h a e m i c A l lodyn ia in M i c e 88 3.3.1. T h e Ef fects of S y s t e m i c Adminis t rat ion of A n a l g e s i c Drugs on Tourn iquet - Induced Pos t -i s c h a e m i c A l lodyn ia in M i c e 88 3.3.1.1. Morph ine (5 mg/kg , 10 mg/kg , 20 mg/kg) 88 3.3.1.2. L idoca ine (75 mg/kg) , Q X - 2 2 2 (140 mg/kg) , and Bup ivaca ine (50 mg/kg) 92 3.3.1.3. Pentobarb i ta l (30 mg/kg , 40 mg/kg) 95 3.3.1.4. R o c u r o n i u m (0.7 mg/kg) 96 3.3.2. T h e Ef fects of S y s t e m i c Admin is t ra t ion (with tail O c c l u d e d by a Tourniquet) of A n a l g e s i c Drugs on Tourn iquet - Induced P o s t - i s c h a e m i c A l lodyn ia in M i c e 99 3.3.2.1. Morph ine (10 mg/kg , 20 mg/kg) 100 3.3.2.2. L idoca ine (75 mg/kg) 104 3.3.3. T h e Ef fects of Per iphera l Adminis t rat ion of S tandard A n a l g e s i c Agen t s on Tourn iquet -Induced P o s t - i s c h a e m i c A l lodyn ia 106 3.3.3.1. Morph ine (1.5%) 106 3.3.3.2. L idoca ine (1%), Bup i vaca ine (0.3%), and Q X - 3 1 4 (2%) 107 3.4. P h a s e IV: Ef fects of S tandard A n a l g e s i c Agen t s on the Deve lopmen t of Tourn iquet - Induced P o s t - i s c h a e m i c A l lodyn ia in M i c e 111 3.4.1. T h e Ef fects of S y s t e m i c Adminis t rat ion of A n a l g e s i c A g e n t s on the Deve lopmen t of Tourn iquet - Induced P o s t - i s c h a e m i c A l lodyn ia in M i c e 111 3.4.1.1. Morph ine (10 mg/kg , 40 mg/kg) and L idoca ine (75 mg/kg) 111 3.4.1.2. A S A (400 mg /kg , 300 mg /kg , and 100 mg/kg) 117 3.4.2. T h e Ef fects of Inhalation G e n e r a l Anaes the t i c s - Isof lurane - on the Deve lopmen t of Tourn iquet - Induced P o s t - i s c h a e m i c A l lodyn ia in M i c e 122 3.4.3. T h e Ef fects of Per iphera l Adminis t rat ion (Intravenous Injection in the Tail) of A n a l g e s i c A g e n t s on the Deve lopmen t of Tourn iquet - Induced P o s t - i s c h a e m i c A l lodyn ia in M i c e . . 126 3.4.3.1. Morph ine (1.5%), L idoca ine (1%), Bup ivaca ine (0.3%) and Q X - 3 1 4 (2%) 127 3.4.3.2. A S A (2.4%) 134 3.4.4. T h e Ef fects of L idoca ine Nerve B lock on the Deve lopmen t of Tourn iquet - Induced Pos t -i s c h a e m i c A l lodyn ia in M i c e 136 vii 4. D I S C U S S I O N 138 4 .1 . T h e Me thod : Tourn iquet - Induced P o s t - l s c h a e m i c A l lodyn ia 138 4 .1 .1 . Impl icat ions f rom the T i m e - C o u r s e of Tourn iquet - Induced P o s t - l s c h a e m i c A l lodyn ia ... 138 4.1 .2 . S t rengths and W e a k n e s s e s of Tourn iquet - Induced P o s t - l s c h a e m i c A l lodyn ia in M i c e . . 140 4 .1 .2 .1 . S t rengths 140 4.1 .2 .2 . W e a k n e s s e s 142 4.2. P h a r m a c o l o g y 143 4 .2 .1 . Re la t ionsh ip be tween ana lges i c and al lodynia b lock ing d o s e s 143 4 .2 .1 .1 . Morph ine 143 4.2 .1 .2 . L idoca ine 145 4 .2 .1 .3 . Bup ivaca ine 146 4.2.1.4. Q X - 2 2 2 / Q X - 3 1 4 147 4 .2 .1 .5 . Pentobarb i ta l 149 4.2.1.6. R o c u r o n i u m . 1 5 0 4.2 .2 . H o w do the ef fects of the ana lges i c agents on prevent ion, deve lopment , and durat ion of tourn iquet- induced pos t - i schaemic a l lodynia c o m p a r e ? 151 4 .3 . Impl icat ions 154 4 .3 .1 . A n i m a l C a r e 154 4.3 .2 . M e c h a n i s m of Tourn iquet - Induced P o s t - l s c h a e m i c A l lodyn ia 154 4 .3 .2 .1 . Cent ra l Si te: Effect of Isof lurane and Morph ine 154 4.3.2.2. Per iphera l : L idoca ine , Bup ivaca ine , and Q X 3 1 4 156 4.4. Comp l i ca t i ons 159 4 .4 .1 . C a n this mode l be appl ied to other s p e c i e s ? 159 4.4 .2 . Cent ra l act ing s i tes for l idocaine and other local anaes the t i cs 160 4.5 . C o n c l u s i o n .....161 4 .5 .1 . M e c h a n i s m of P o s t - l s c h a e m i c A l lodyn ia 161 4.5 .2 . Potent ia l of the Me thod 163 4 .5 .3 . Future A v e n u e s for R e s e a r c h 164 viii Bibliography 166 ix LIST OF FIGURES Figure 1: T h e appara tus used in the exper iment 29 F igure 2: A m o u s e with a tourniquet appl ied around the b a s e of its tail 30 F igure 3: P inpr ick test 31 F igure 4: V o n Frey hair test 32 F igure 5: T h e relat ionship be tween the log ( latency of a p p e a r a n c e of pos t - i schaemic al lodynia) and log (duration of i schaemia ) 48 F igure 6: T i m e - c o u r s e of tourn iquet- induced pos t - i schaemic a l lodynia at posi t ions D and P (first 300 minutes after the tourniquet w a s re leased) 51 F igure 7: T i m e - c o u r s e of tourn iquet- induced pos t - i schaemic a l lodynia at pos i t ions D and P (6-day fol low-up period) 55 F igure 8 T h e magn i tude of tourniquet- induced pos t - i schaemic a l lodynia in m ice 61 F igure 9 T h e recovery rates of tourn iquet- induced pos t - i schaemic a l lodynia in m ice 65 Figure 10: T h e effect of i s c h a e m i a on pinprick r e s p o n s e at posi t ion D 67 Figure 11: Ef fects of s u b c u t a n e o u s inject ions of morph ine (at the back of the neck) on the pinprick r e s p o n s e of m ice 71 F igure 12: T h e ef fects of intraperi toneal injection of l idocaine (75 mg/kg) on the pinprick r e s p o n s e s of m ice 75 Figure 13: T h e effect of intraperi toneal injection of (a) 40 mg/kg and (b) 75 mg/kg l idocaine on the per fo rmance of m ice on a rotarod 76 Figure 14: T h e effect of s u b c u t a n e o u s injection (at the back of the neck) of 75 mg/kg l idocaine on normal m ice 77 Figure 15: T h e effect of s u b c u t a n e o u s injection (at the back of the neck) of Q X - 2 2 2 (140 mg/kg) on normal m ice 78 Figure 16: T h e ef fects of intraperi toneal injection of pentobarbi ta l (30 mg/kg , 40 mg/kg) on (a) the pinprick r e s p o n s e and (b) the per fo rmance on a rotarod of the m ice 80 F igure 17: T h e ef fects of intraperi toneal injection of l idocaine (75 mg /kg , 0.5 ml) immediate ly after a tourniquet w a s app l ied on normal m ice 84 Figure 18: T h e ef fects of s u b c u t a n e o u s injection (at the back of a m o u s e neck) of var ious d o s e s of morph ine on the tourn iquet- induced pos t - i schaemic a l lodynia in m ice 90 Figure 19: T h e ef fects of s u b c u t a n e o u s injection (at the back of a m o u s e neck) of (a) 75 mg/kg l idocaine and (b) bup ivaca ine (50 mg/kg) on tourn iquet- induced pos t - i schaemic a l lodynia in m ice 94 Figure 20 : T h e ef fects of intraperi toneal injection of pentobarbi tal (30 mg/kg , 40 mg/kg) on (a) tourniquet-induced pos t - i schaemic a l lodynia (at posit ion D) in m ice and (b) their pe r fo rmance on a rotarod 97 x Figure 21 : T h e effect of intraperi toneal injection of rocuron ium (0.7 mg/kg) on the pe r fo rmance on rotarod of a l lodynic m ice 98 Figure 22: (a, b) T h e ef fects of ip injection (with the b lood f low to the tails occ luded) of morph ine (10 mg/kg , 20 mg/kg) on tourniquet- induced pos t - i schaemic a l lodynia in m ice 102 F igure 23 : T h e ef fects of l idocaine (75 mg/kg , ip) (with b lood f low to the tails occ luded) on tourniquet-induced pos t - i schaemic a l lodynia in m ice 105 Figure 24 T h e ef fects of per ipheral local inject ions of (a) morph ine (1.5%), (b) l idocaine (1%) and (c) bup ivaca ine (0.03%) on tourn iquet- induced pos t - i schaemic a l lodynia in m ice 109 Figure 25 T h e ef fects of intraperi toneal inject ions of morph ine (10 mg/kg) , l idocaine (75mg/kg) and sa l ine control (0.5ml) on the deve lopment of a l lodynia in m ice 114 Figure 26 T h e ef fects of morph ine (40 mg/kg) and l idocaine (75 mg/kg) on the deve lopmen t on tourniquet-induced pos t - i s chaem ic a l lodynia in m ice 116 Figure 27 T h e ef fects of var ious d o s e s of A S A (100 mg/kg , 300 mg /kg , and 4 0 0 mg/kg) on the deve lopment of tourn iquet- induced pos t - i schaemic a l lodynia in m ice 119 Figure 28 T h e ef fects of genera l anaes the t i cs on the deve lopmen t of tourn iquet- induced pos t - i schaemic a l lodynia in m ice 124 F igure 29: T h e ef fects of per ipheral injection of morph ine (0.05ml, 1.5%) on the deve lopment of tourn iquet- induced pos t - i schaemic a l lodynia in m ice 130 F igure 30: T h e ef fects of in t ravenous injection (distal to the tourniquet-si te) of l idocaine (0.05 ml, 1%) into the m o u s e tails on the deve lopment of tourn iquet- induced pos t - i schaemic a l lodynia at posi t ions D and P of the tails 131 F igure 31 : T h e effect of in t ravenous injection (distal to the tourniquet-site) of bup ivaca ine (0.05 ml, 0.03%) into the m o u s e tails on the deve lopment of tourn iquet- induced pos t - i schaemic a l lodynia at posi t ions D and P of the tails 132 Figure 32 T h e effect of in t ravenous injection (distal to the tourniquet-site) of Q X - 3 1 4 (2%, 0.05 ml) into the m o u s e tails on the deve lopmen t of tourn iquet- induced pos t - i schaemic a l lodynia at posi t ions D and P of the tails 133 F igure 33: T h e ef fects of in t ravenous injection (distal to the tourniquet-site) of A S A (0.05 ml, 2.4%) into the m o u s e tails on the deve lopment of a l lodynia at posi t ions (a) D and (b) P of the tails 135 Figure 34: T h e effect of l idocaine (2%, 0.03 ml) induced nerve block on the deve lopment of post-i s c h a e m i c a l lodynia in m ice 137 xi LIST OF ABBREVIATIONS A S A Ca C N S "P iv K L sc mg mg/kg ml n= Na NMDA NSAIDs P N S r.p.m. S E M - acetylsalicylic acid - calcium - central nervous system - intraperitoneal - intravenous - potassium - litres - subcutaneous - milligram - milligram per kilogram weight - millilitres - sample size - sodium - N-methyl-D aspartate - non-steroidal anti-inflammatory drugs - peripheral nervous system - rotation per minute - standard error of the mean V F H - von Frey hair WDR neuron - wide dynamic range neuron 1. I N T R O D U C T I O N 1.1. Brief Introduction to Studying Tourniquet-Induced Post-ischaemic Allodynia in Mice The goal of studying pain in experimental animals is to explore the phenomenon of pain itself (Dunber, 1989). According to Dubner (1989), "a major purpose of such studies is to provide knowledge that can ultimately be applied to the management of acute and chronic pain conditions in human and animals". Currently available methods for investigating allodynia and hyperalgesia are generally invasive. Toxic chemicals such as capsaicin, formalin, nociceptin (Calo, 1998) and mustard oil (Cervero and Laird, 1996) are often injected into experimental animals to induce hyperalgesia and allodynia. Even though the injection of these substances can induce allodynia and hyperalgesia, the underlying mechanisms of the allodynia induced by these methods and that normally observed in humans could be quite different from one another. Moreover, the spinal nerve (L5 and L6) ligation method developed by Kim and Chung (1991) required irreversible surgical procedures to be performed on the experimental animals. Compared to these methods descried, the application of a tourniquet around the base of a mouse tail can induce allodynia in a reversible, rapid, convenient, and less invasive way. In our study, a tourniquet was applied around the base of a mouse tail in order to induce ischaemia at the distal extremities. This method has been applied to other animals, such as rats (Gelgor et al., 1986a), for inducing hyperalgesia and allodynia. The post-ischaemic hyperalgesia and allodynia induced in those studies were found to 1 be reproducible (Gelgor et al., 1986a, Chabel et al., 1990; Kupers et al., 1998; Crews and Cahall, 1999). We investigated tourniquet-induced post-ischaemic allodynia in mice with respect to its time course, and its mechanism of development. Previous experiments done in our laboratory had demonstrated that the application of a 60-minute tourniquet around the base of a mouse tail was effective in inducing allodynia in the tail. Mechanical allodynia/hyperalgesia were often reported qualitatively rather than quantitatively. In order to describe the tourniquet-induced post-ischaemic allodynia in a quantitative way, we investigated its time course, including its onset and overall duration. Consequently, the actions of various drugs could be quantified with respect to their actions on a specific phase of post-ischaemic allodynia. The first set of the experiments determined the characteristic of the post-ischaemic allodynia: the relationship between various duration of tourniquet application and the time-course of the induced post-ischaemic allodynia. We also tested the effects of a few standard analgesic agents on tourniquet-induced post-ischaemic allodynia in mice in order to demonstrate how our method could be applied to the study of the pharmacology and the mechanisms associated with tourniquet-induced post-ischaemic allodynia. Besides the advantage described above, the method used in our experiments allowed the investigation of the mechanism of development of allodynia. An advantage of applying a tourniquet around the base of a mouse tail was that the blood flow to the tail (distal to the tourniquet-site) was stopped. In other words, the blood trapped in tail was technically isolated from the systemic circulation. Various analgesic and anaesthetic agents could be injected either systemically into the animal or peripherally 2 into the tail. The role of specific nerve fibres (nociceptive fibres and touch fibres) and the receptors involved in the development of post-ischaemic allodynia, the interactions between the central and peripheral nervous system, and the site at which the sensitisation process might have taken place could potentially be determined from the responses of the mice to the actions of the injected analgesic and anaesthetic agents. The method that we have developed allowed the quantification of tourniquet-induced post-ischaemic allodynia in mice with respect to its time course. It also allowed the investigation of potential treatments for, and the underlying mechanisms of, tourniquet-induced post-ischaemic allodynia in a convenient, less invasive, and relatively inexpensive way. 1.2. Definitions 1.2.1. Taxonomy of Pain Pain is a subjective unpleasant sensation resulting from the stimulation of nociceptive nerve endings by injury, disease, or other harmful factors (Hyman and Cassem, 1994). According to Dorland's Illustrated Medical Dictionary 28 th edition, pain is defined as "a more or less localized sensation of discomfort, distress, or agony, resulting from the stimulation of specialized nerve endings." The taxonomy committee of the International Association for the Study of Pain (1979) considered the various definitions of pain and concluded that pain is "an unpleasant sensory and emotional experience associated with actual or potential tissue damage, or described in terms of such damage" (Wall, 1989). Each individual learns about the application of the word "pain" via experiences in early life. 3 Nociception, on the other hand, refers to the neural mechanisms by which noxious stimuli are detected. Activating the nociceptive pathway usually leads to the experience of pain; however, there are many other factors, such as stress, past experiences, and psychological states, that can affect the perception of pain (Wall, 1989). Pain can be generally classified into acute pain and chronic pain. Acute pain is often perceived when the nociceptors are activated, resulting in firing of the nociceptive nerve fibres. The pain perceived is usually sharp and transient. For example, pain that results from a pinprick can be classified as acute pain. On the other hand, chronic pain refers to a continuous discomfort that lasts for a long period of time, from a few hours to several weeks. An example of chronic pain is muscle pain after intensive exercise. Chronic pain is sometimes associated with neuronal damage in the central nervous system (CNS) and/or the peripheral nervous system (PNS). In patients who are experiencing chronic pain, nociceptive fibres fire spontaneously or at a lowered threshold. In some cases, non-noxious stimuli, such as a gentle touch, can induce pain or exaggerated discomfort ("allodynia"). The excitability of nerve cells is changed, such that previously ineffective inputs become effective (Wall, 1989). Both central and peripheral mechanisms of nerve sensitisation for the development of chronic pain have been proposed, but the exact mechanism of the development of chronic pain remains to be investigated. 4 1.2.2. Taxonomy of Allodynia and Hyperalgesia Allodynia is defined as the perception of pain induced by a normally innocuous stimulus, such as a gentle touch. It is to be distinguished from hyperalgesia, which is characterized by a lowered pain threshold and an augmented pain to normally painful stimuli (LaMotte, 1991). Lewis (1936) was a pioneer in the study of hyperalgesia. According to his findings, hyperalgesia can be sub-divided into primary hyperalgesia and secondary hyperalgesia. Primary hyperalgesia normally occurs at the site of injury, whereas secondary hyperalgesia occurs in normal and undamaged surrounding tissue (Lewis, 1936). 1.3. Background 1.3.1. Pain, Allodynia and Hyperalgesia Pain is one of the most common phenomena we experience. Without considering its scientific definition, pain is an unpleasant feeling that we tend to avoid. According to Wall (1989), "pain is recognized when there is an abnormal reaction (behaviour, muscle contraction, autonomic response, endocrine response etc.) which becomes less abnormal when an analgesic procedure is introduced". Sometimes, it can also be interpreted as an unpleasant feeling that may affect people's work performance, mood, or daily activities. However, the sensation of pain also has its practical importance, in spite of its unpleasantness. Our bodies are so fragile that they can be damaged easily. When there is a potential hazard, the sensation of pain can alert the individual to escape from the hazard. Moreover, when part of the body is damaged, for example an arm is broken, the sensation of pain can protect the damaged part from 5 further damage by preventing normal movement. The practical importance of pain is to protect the damaged parts as well as to alert the individual to escape from harmful stimuli. Although the practical importance of pain is to protect an individual from hazards, in some cases where the apparent causes of pain are unknown or when pain becomes chronic, pain loses its practical meaning. This type of pain is often described as pathological pain (or chronic pain), which is often associated with nerve damage (Woolf, 1991). The treatments for many types of pathological pain are currently inadequate. According to Wall (1989), problem pains can be grouped into three major classes: 1) Pain with apparent cause but inadequate treatment 2) Pain with unknown cause but adequate treatment 3) Pain with unknown cause and inadequate treatment. There is a need for studying pain as long as there are pains which we do not understand and which are inadequately treated (Wall 1989). The development of treatments for relieving the pain problems listed above can be achieved in two ways: (1) through the development of new drugs from the structures of currently effective analgesic drugs, and (2) through the investigation of mechanisms of the pathological pain to determine a specific target for which new analgesic agents can be developed. Allodynia and hyperalgesia are two aspects of chronic pain of which the understanding of their mechanisms and the available treatments are currently inadequate. It is known that tissue injury causes hyperalgesia, which is the perceptual companion of inflammation (Wall 1989). Lewis (1936) had divided hyperalgesia into two 6 categories: primary hyperalgesia and secondary hyperalgesia. The former one occurs at the site of injury, and the latter one occurs in the surrounding undamaged area. Two separate mechanisms have been proposed to explain the spread of hyperalgesia, both which differ in their emphasis on the importance of the peripheral and central nervous system (Coderre and Melzack, 1987). The current understanding of allodynia and hyperalgesia will be further discussed in later sections (1.4.2). 1.3.2. Tourniquet pain A tourniquet is a device for compressing blood vessels by application around an extremity in order to prevent the flow of blood to or from the distal area. There are various kinds of tourniquets, which are named chiefly after their inventors. Pain has often been reported in human patients after surgical procedures involving the use of pneumatic tourniquets. The tourniquet is used to occlude blood flow to a limb for various surgical procedures at the distal extremities. However, a severe, dull, aching sensation at the tourniquet-site and distal extremity are often reported after the surgery. This type of pain is usually referred to as tourniquet pain (Crews et al., 1991; Crews, et al., 1994). The mechanisms of the development of tourniquet pain are still unknown, which hinders the development of specific drugs for the problem. The application of a tourniquet around a limb/tail of an animal, such as a rat, was found to be effective in inducing post-ischaemic hyperalgesia and allodynia to both mechanical and thermal stimuli. Post-ischaemic hyperalgesia to noxious thermal stimuli in a rat tail after the application of a 60-minute tourniquet at the base of the tail has been reported (Gelgor, et al., 1986a). Photochemically induced ischaemia at a rat's 7 sciatic nerve was also found to induce a dose-dependent and highly reproducible mechanical and thermal allodynia (Kupers et al., 1998). The development of the current method for investigating tourniquet-induced post-ischaemic allodynia in mice provided valuable information about the mechanism of and the potential treatment for allodynia through simple, rapid and convenient experimental procedures. Moreover, allodynia was induced in the mice without injecting any toxic chemicals or performing any irreversible surgical procedures. 1.3.3. Methods for evaluating allodynia and the von Frey hairs Currently, there are various ways of testing for allodynia in experimental animals and humans, for example, immersing the part of the body in warm water, and stroking the skin with a soft paintbrush. Von Frey filaments were chosen in these experiments to test for the presence of allodynia because of their ease of application and standardization; the forces exerted by the von Frey filaments are known and effectively constant, which enhanced the reproducibility of the results. On the other hand, thermal allodynia is less readily evaluated; unless radiant or laser-generated types of heat sources are used in the investigation of thermal allodynia, conducted heat, which requires direct contact between the heat source and the skin of the animal, can lead to stimulation of the mechanoreceptors, thus reducing specificity of the heat stimulus (Murrin and Rosen, 1985). According to review by Weinstein et al. (1996), the von Frey filaments used in these experiments were first described by Max von Frey at the beginning of the 20 t h century. He discovered that the forces exerted by horse hairs were not proportional to 8 their magnitudes of bending. In other words, the magnitude of the force does not obey Hooke's Law (proportionality between force and the change in length of a spring). The force exerted on a surface by such a piece of hair was found to be fairly constant. Von Frey used those hairs to explore tactile sensation. In our study of tourniquet-induced post-ischaemic allodynia, the presence of allodynia in a mouse tail was assessed by touching the tail with a piece of nylon von Frey filament. Commonly used monofilament is a single strand of nylon, which is able to induce a characteristic downward force that does not depend on the bending magnitude of the monofilament when it is slightly bent on a surface. The actual downward force of contact increases linearly until the monofilament slightly bends. At this point, the actual force varies around the characteristic force for that monofilament. Further bending of the filament does not increase the induced force. Equations predict the characteristic force from the diameter and the length of the monofilament rather than from the degree of bending. The material of the filaments also affects the resulting downward forces. The terms "von Frey hairs" (VFH) and "von Frey filaments" are both current and these terms are used interchangeably in this thesis. The term "von Frey hair test" was defined as the test for the presence of allodynia in a mouse tail by touching the tail with a piece of V F H , and observing whether or not this was followed by a tail flick. 9 1.4. Present Understanding about the Mechanisms of Pain, Allodynia and Hyperalgesia 1.4.1. Present Understanding about Nociception 1.4.1.1. Receptor Level Nociceptors, or pain receptors, respond to stimuli that potentially or actually produce damage. Nociceptors can be classified into 2 major groups: A-8 mechanical nociceptors and C-polymodal nociceptors (Wall, 1989; Hyman and Cassem, 1994). According to Berne (1996), the main difference between these 2 different types of nociceptors is that A-5 mechanical nociceptors are supplied by finely myelinated (or A-8) axons, whereas C polymodal nociceptors are supplied by unmyelinated (or C) fibres. A-8 mechanical nociceptors respond to strong mechanical and thermal stimuli and they normally do not respond to noxious chemical stimuli unless they have been previously sensitized. On the other hand, C polymodal nociceptors respond to several types of noxious stimuli, such as mechanical, thermal, and chemical. 1.4.1.2. Ch emical Mediators According to the review by Hyman & Cassem (1994), during the inflammatory process, damaged tissues normally release various chemical mediators that are capable of activating the nociceptors. K + and H + produce by the immune system may also activate nociceptors. During an inflammation, platelets release serotonin and mast cells release histamine at the site of injury. These inflammatory products can induce the 10 production of bradykinin from kininogen. Bradykinin was found to be one of the most potent activators of nociception. Prostaglandins and leukotrienes, which are also released from damaged tissue, play important roles in the development of hyperalgesia and allodynia. They do not directly activate nociceptors; instead, they lead to sensitization of all classes of primary nociceptive afferents (Hyman & Cassem, 1994). Sensitization of the primary nociceptive afferents may involve (1) an increased or prolonged firing to suprathreshold stimuli, and (2) an increase in spontaneous firing of the pain fibres. (Levine, 1993; Hyman & Cassem, 1994) 1.4.1.3.Neuronal Level The axons that mediate nociception are mainly A-5, and C fibres (Wall, 1989). The classification is mainly based on their diameters and degrees of myelination (Wall, 1989). A - 5 fibres are thinly myelinated with axon diameters of 2-5 um, and they are responsible for transmitting mechanical and thermal pain sensation (Strichartz, 1988). On the other hand, C fibres are unmyelinated nerve fibres with axon diameters of 0.2 to 1.5 u.m. They are also responsible for transmitting mechanical and thermal pain sensation (Strichartz, 1988). Because of their lack of myelination, C fibres conduct very slowly at a speed of less than 2 m/s (approximate 1/10 of that of A- S fibres) (Hyman & Cassem, 1994). Consequently, there are usually two phases of pain sensation. The first phase of pain sensation, called the "first pain" (mediated by A - 8 fibres), is sharp, rapid and brief. It is then followed by a second phase of pain sensation, called the "second 11 pain" (mediated by C fibres), which is a burning and more prolonged unpleasant sensation (Strichartz, 1988). Other related fibres are the A-p fibres, which innervate muscles and transmit touch and pressure sensations (Strichartz, 1988). Their axon diameters are 5-12 pm and their conduction speed is about 30-70 m/s). The potential roles of these fibres in the development and perception of allodynia remain to be investigated. 1.4.1.4.CNS Level A- 5 and C fibres enter the spinal cord at the lateral division of the dorsal root (rev. Hyman and Cassem, 1994); they then bifurcate and either ascend or descend in Lissauer's tract (the dorsolateral fasciculus) for one to three spinal segments. These fibres then synapse in the dorsal horn of the spinal cord on neurons in laminae I, II, and V of the grey matter. However, fibres from the face and head synapse in the pars caudalis of the trigeminal nucleus. The grey matter of the spinal cord is divided into 10 laminae, and the dorsal horn is composed of Laminae I to VI (rev. Hyman & Cassem, 1994). The primary nociceptive afferents terminate on the excitatory and inhibitory interneurons. They also terminate on the projection neurons that relay the nociceptive information to the brain stem and thalamus through various pathways. The excitatory transmitters released by the incoming sensory afferents are not entirely known, but the amino acid glutamate and substance P appear to be involved in nociception. Central nociceptive neurons at the level of the dorsal horn and higher (e.g. thalamus and cerebral cortex) have two different types of responses to the stimulation 12 of their receptive fields. Some of these neurons, termed nociceptive-specific neurons, have higher thresholds. Wide dynamic range (WDR) neurons, on the other hand, receive inputs from both nociceptive and non-nociceptive afferents. Within the dorsal horn, lamina I consists of mainly high-threshold neurons, but there are also some W D R neurons. On the other hand, lamina V has a higher proportion of W D R neurons. Excitatory interneurons, associated with the nociceptive afferents, in the dorsal horn are involved in the spinal withdrawal reflex. On the other hand, inhibitory interneurons within the dorsal horn are involved in blocking nociception, i.e. inducing analgesia. Some of these inhibitory neurons, which contain endogenous opioid peptides, can be activated locally by incoming sensory afferents and the descending analgesic pathways, resulting in inhibition of nociception at the dorsal horn level. Cells on the dorsal horn can be classified in at least five different ways (Wall, 1989): 1) Laminar organization; 2) Cell shape; 3) Chemistry; 4) Destination of axons; 5) Brief response to brief stimuli. However, as emphasized by Wall (1989), a fixed and rigid labelling of cells is inappropriate, since the physiological characteristics of a cell can be changed with time, particularly during pathological circumstances. The following three mechanisms that can possibly change the excitability of dorsal horn cells have been proposed: (1) gate control, (2) sensitivity control, and (3) connectivity control. 13 In the development of allodynia and hyperalgesia, it has been proposed that the dorsal horn cells are changed into a pathological state of increased excitability. In addition, it has been suggested that pain is triggered not only because of the excitation by nociceptive afferents, but also because of excitation from normal low-threshold afferents (such as A-p fibres). However, the underlying mechanism has not yet been identified (Wall, 1989). 1.4.1.5.Psychological Factors The pain induced by tissue injury can be considered in terms of two components: "a specific intensity-related component that maps the pain report in terms of the intensity and location of an effective stimulus (sensory-discriminative), and a component of perceptual processing defining environmental context and complex behavioural attributes such as emotion and anxiety (affective-motivational)" (Yaksh 1997). The neurological dimensions of pain were described in the previous sections. What are the psychological dimensions of pain? According to Bellissimo & Tunks (1984), one good example that introduces psychological dimensions into pain study is the phenomenon of placebo response. Chronic pain has been suggested as a form of depression. The correlation between chronic pain to depression has been described by many researchers. Bellissimo & Tunks (1984) proposed, "It is intuitively obvious to assume that the psychological distress associated with chronic pain is the result of the 'stress' of that pain". Also, it has been shown that pain experience can be altered by changes in the thought patterns 14 and expectations of an individual. As demonstrated in the human cases above, psychological factors could be involved in pain perception. Unfortunately, the magnitudes of the psychological factors are hard to quantify in human as well as in animal studies. 1.4.2. Present understanding of allodynia and hyperalgesia Lewis (1936) studied the process by which a large area of undamaged skin surrounding a local cutaneous injury can become hyperalgesic to mechanical stimuli. Hyperalgesia is characterized by lowered pain thresholds and increased pain intensity to normally painful stimuli (Lewis, 1936). It is observed at the site of injury (primary hyperalgesia) as well as the surrounding normal, undamaged tissues (secondary hyperalgesia) (Lewis, 1936). Within the area where primary/secondary hyperalgesia was observed, a normally innocuous stimulus, such as a gentle touch, could evoke soreness or tenderness, and a normally painful prick from a needle could induce amplified and prolonged pain. Technically, by the definition of hyperalgesia and allodynia (section 1.2.2), both hyperalgesia and allodynia were observed within the area of "primary/secondary hyperalgesia" described by Lewis. Lewis proposed that the spread of hyperalgesia to surrounding undamaged tissue was mediated by peripheral nerves. However, Hardy et al. (1950) proposed that a central mechanism could also be involved. 15 1.4.2.1.Peripheral mechanisms In the experiments of Lewis (1936), he showed that spread of secondary hyperalgesia induced by mechanical stimulation could be blocked by locally anaesthetizing the skin surrounding the site of injury, with 1% novocaine, suggesting that a peripheral mechanism could be involved in the development of secondary hyperalgesia (and allodynia) (Lewis, 1936). Subsequently, various local factors that at least hypothetically contribute to the development of hyperalgesia and allodynia have been proposed, including changes in pH values, recruitment of "sleeping receptors", and direct sensitisation of peripheral nerve fibres by products of inflammation. According to Handwerker and Reeh (1992), even though A-p and A-5 units were found to be neither excited nor sensitised by acidic pH levels, polymodal or mechano-heat responsive units (C fibres) demonstrated irregular low-frequency discharges with poor response characteristics in acidic conditions. Moreover, repeated or prolonged treatment of the C-fibres isolated from rats with low pH was found to be effective in inducing a significant and sustained decrease of the mechanical thresholds in those C fibres. Besides acidic pH, injection of capsaicin has also been shown to induce allodynia in humans as well as rats. In a study with human subjects, anaesthetizing the skin prior to the injection of capsaicin was found to be effective in preventing the spread of secondary hyperalgesia (allodynia and hyperalgesia) (LaMotte et al., 1991), suggesting a peripheral mechanism could be involved in the development of secondary hyperalgesia. Schmidt et al. (1994) proposed the existence of "sleeping receptors", which could 16 be recruited during inflammation, resulting in hyperalgesia and allodynia. Normally inactive C fibres were shown to be "awakened" by the induced inflammatory conditions (Schmidt et al., 1994; Maclver and Tanelian, 1992, Devor, 1991). This piece of evidence also suggested a peripheral mechanism could be involved. There are many endogenous substances that have been shown to be effective in decreasing pain threshold via different means of measurement, and are therefore candidates as inducers of hyperalgesia. The following have been shown to reduce paw pressure withdrawal thresholds in rats when administered peripherally: bradykinin, serotonin (5-HT), prostaglandins (PGs), leukotrienes, norepinephrine, adenosine, neurokinin A , substance P, interleukins, tumor necrosis factor a, cAMP, and nerve growth factor (Reeh et al., 1997). However, to what extent these substances are actually involved in the development of hyperalgesia (and allodynia) remains to be elucidated. 1.4.2.2.Central mechanisms Various studies have indicated that the central nervous system is involved in the development of hyperalgesia and allodynia (Hardy et al., 1950; LaMotte et al., 1992; Torebjbrk et al., 1992; Sang et al., 1996; Kaneko et al., 1997; Stubhaug et al., 1997) Gamma-aminobutyric acid ( G A B A ) is one of the major neurotransmitters in the central nervous system. The importance of G A B A in the modulation of nociceptive transmission has been demonstrated (Kaneko et al., 1997). Intrathecal administration of G A B A has been shown effective (via G A B A A receptors) in inducing anti-nociceptive 17 action to acute pain stimulus (Kaneko et al., 1997). Intrathecal injection of high doses of G A B A A receptor antagonists was found to be effective in inducing mechanical allodynia and spontaneous pain (Kaneko et al., 1997). Spinally administered G A B A A receptors antagonists were also found to enhance the response of dorsal horn neurons to the activation of A-(3 fibres, which were responsible for the touch sensation (Kaneko et al., 1997). Ito et al. (1997), on the other hand, have shown the importance of NMDA (N-methyl-D aspartate) receptors in the development of allodynia induced by intrathecal injection of P G E 2 , in mice. The injection of MK-801, an NMDA receptor antagonist, was found to be effective in inhibiting PGE 2 - induced allodynia. 1.4.3. Currently used standard analgesic agents and their mechanisms of actions 1.4.3.1.Opioid Analgesics According to the review by Reisine and Pasternak (1996), morphine is a standard opioid analgesic agent that is widely used in human patients and experimental animals. There are now many compounds with pharmacological properties similar to those of morphine, but none has proven to be clinically superior in relieving pain. Morphine remains the standard against which new analgesics are measured. Opioid analgesics also provide symptomatic relief of pain, cough, or diarrhea, but (in human beings) they also can induce drowsiness, changes in mood, and mental clouding. When therapeutic doses of morphine are given to patients with pain, they usually report a reduction in pain intensity. 18 It is believed that opioid-induced analgesia is mainly due to actions at several sites within the C N S . In brief, morphine and related opioids induce their major effects on the central nervous system and the gut through p-receptors. Although morphine is relatively selective for u-receptors, it can interact with the other opioid receptors (K and 8 receptors) at higher doses. (Reisine and Pasternak, 1996; Rang et al., 1995) According to Reisine and Pasternak (1996), at least three mechanisms of actions for opioids have been proposed. Opioid receptors on the central terminals of primary afferent nerves mediate inhibition of the release of neurotransmitters, including substance P. Morphine also exerts post-synaptic inhibitory actions, either indirectly by activating K + conductance or directly by decreasing the C a 2 + conductance, on interneurons and on the output neurons of the spinothalamic tract that transmits nociceptive information to higher centres in the brain. Analgesia can also be induced by the instillation of morphine into the third ventricle or in various sites in the midbrain and medulla. 1.4.3. l.Local Anaesthetics Local anaesthetics reversibly block the action potentials responsible for nerve conduction if they are administered locally to nerve tissues in appropriate concentrations (Catterall and Mackie, 1996, Rang et al., 1995). A practical advantage of the local anaesthetics is that their action is reversible at clinically relevant concentrations without resulting in noticeable damage to the nerves (Catterall and Mackie, 1996). In brief, local anaesthetics block conduction by decreasing or preventing 19 the large transient increase in the permeability of excitable membranes to sodium ions (Na +). The local anaesthetics selected in this study were lidocaine, bupivacaine. Two local anaesthetic analogues were also tested: QX-222 and QX-314. Lidocaine (xylocaine), introduced in 1948, is now one of the most widely used local anaesthetics (Catterall and Mackie, 1996, Rang et al., 1995). Pre-injury (local) infiltration with lidocaine has been found to be effective in reducing hyperalgesia and allodynia in human volunteers (Dahl, 1993; Wallace et al., 1997). Analgesic properties of lidocaine when administered systemically have also been reported (Boas et al., 1982; Ferrante et al., 1996). For example, intravenous infusion of lidocaine (200 to 400 mg infused over 30 minutes) in human patients was found to be effective in relieving chronic pain in these patients through an undetermined mechanism of action (Petersen et al., 1986). Prolonged relief of tactile allodynia by intravenous injection of lidocaine in neuropathic rats has also been reported (Chaplan etal . , 1995). Bupivacaine, introduced in 1963, is another widely used amide local anaesthetic (Catterall and Mackie, 1996). It is a potent local anaesthetic that is capable of inducing prolonged anaesthesia. Moreover, it has been shown that bupivacaine is more selective for the sensory fibres than the motor fibres (Catterall and Mackie, 1996). QX-222 and QX-314 are compounds that are positively charged quaternary derivatives of lidocaine and are impermeable through normal biological membranes and blood-brain barrier (BBB) (Zapata et al., 1997). In previous experiments performed in our laboratory, QX-222 and QX-314 were found to act like lidocaine and bupivacaine in that they induced 20 local anaesthesia, but for relatively prolonged periods. 1.4.3.3. General Anaesthetics/General Depressants The state of general anaesthesia is a drug-induced absence of all sensations (Marshall and Longnecker, 1996). Isoflurane, a general inhalation anaesthetic, was used in this study for two reasons: (1) it could block the perception of pain centrally, which allowed the investigation of the role of conscious perception of pain in the development of post-ischaemic allodynia, (2) it could minimize the suffering of the mice during potentially painful experimental procedures, such as applying a tourniquet around the tail of an allodynic mouse (Marshall and Longnecker, 1996). Isoflurane, introduced in 1981, was chosen in the experiment because it could induce general anaesthesia in mice smoothly and rapidly (Marshall and Longnecker, 1996). Moreover, it allowed a rapid emergence from the induced general anaesthesia (Marshall and Longnecker, 1996). The depth of anaesthesia could also be adjusted rapidly with isoflurane. All these properties have made isoflurane particularly useful in the study, especially for the experiments that required a very brief duration of anaesthesia in mice. 1.4.3.4. Neuromuscular Junction Blocker In the experiment, the tail withdrawal reflex of a mouse was monitored. A positive response to pinprick or touch was indicated by a flick of the tail. It was assumed that if a mouse did not respond in this way to pinprick or touch, the mouse did not 21 perceive any pain. However, some of the drugs used, such as the local anaesthetics and their analogues, could potentially block the motor nerve fibres. If the motor nerve fibres of a mouse were blocked, the negative response (i.e. the lack of response) to pinprick or touch could be due to the inability to respond rather than the analgesia induced by the injected compounds. In other words, the actual analgesic actions of the injected drugs would be masked by the blockade of the motor nerve fibres, resulting in the negative response to pinprick or touch. Therefore, a neuromuscular junction blocker, which does not possess any analgesic properties, was included in our study as a control compound. Rocuronium, an ammonio steroid, is a non-depolarizing neuromuscular junction blocker. The drug acts by binding competitively to cholinergic receptors at the motor end-plate to antagonize the action of acetylcholine, an effect which is reversible by the administration of acetylcholinesterase inhibitor, such as neostigmine and edrophonium (Welbank, 2000). 1.4.3.5.Non-steroidal Anti-inflammatory Drugs (NSAIDs) In the experiment, a tourniquet was used to induce ischaemia in a mouse tail. The build-up of the products of inflammation in the ischaemic area could have sensitised the nerve fibres, resulting in allodynia. Prostaglandin, one of these products of inflammation, could have played an important role in the development of tourniquet-induced post-ischaemic allodynia. Injection of acetylsalicylic acid (ASA), which blocks the production of 22 prostaglandins by blocking the cyclo-oxygenase-1 (COX-1) and - 2 (COX-2) (Rang et al, 1995), has been found to be effective in abolishing post-ischaemic hyperalgesia to noxious thermal stimulus in rats (Gelgor et al, 1986b; Gelgor et al, 1992b). Pre-treatment of the mice with intraperitoneally injected A S A (300 and 400 mg/kg) has also been shown effective in reducing pain-related behavioural response consisting of vigorous biting, licking and scratching of the caudal part of the body, in a dose-dependent manner (Hunskaar et al., 1985). A S A was included in our study of tourniquet-induced post-ischaemic allodynia for investigating the potential role of prostaglandins in the development of allodynia in mice. 23 2. MATERIALS AND METHODS 2.1. Experimental Definition of Pain and Allodynia A mouse normally flicks its tail in response to a "painful stimulus" (such as a pinprick) but not an "innocuous" stimulus (such as a gentle touch). It was assumed that this tail withdrawal response of a mouse is indicative of perception of pain. In the experiment, the tail withdrawal reflex of a mouse was utilized as an indication of nociception. The response of a mouse to a painful stimulus, a pinprick, and a normally innocuous stimulus, the touch of a piece of nylon von Frey filament, was measured. The specifications of these were discussed in the Material and Method section (section 2.2.2 and 2.2.3) in detail. A positive response, which was the evidence for the perception of pain or discomfort by a mouse, was indicated by a flick of the tail upon stimulation by the pinprick device or a von Frey filament. The tail-flick response to the touch of a von Frey filament was taken to indicate the presence of allodynia in the tail and it is referred to as the "response to touch" in this thesis. Drugs that blocked the pinprick response were assumed to have blocked the pain associated with the pinprick; correspondingly, drugs that blocked the tail-flick response to touch of an allodynic mouse were assumed to have blocked the pain (allodynia) associated with the touch. 2.2. Materials 2.2.1 Experimental Subjects Female CD-1 mice (20 - 30 g) obtained from Charles River, Quebec, Canada were used in all experiments. They were kept in groups of 15 in 10 L transparent 24 Plexiglas cages, which were stored in the animal room in the basement of Medical Sciences Building C, UBC, prior to the experiment. There were at least two advantages of using mice in the experiment: 1) They were small and easy to manipulate. 2) They required less drugs as compared to larger animals, which could reduce the cost by a large percentage when expensive new drugs were being tested. In some of the experiments, tourniquet-induced post-ischaemic allodynia in a mouse tail was detected by monitoring the withdrawal response to the touch of a piece of von Frey filament. However, because of the variation in the sensitivity to touch among different mice, some mice might respond to the touch of the von Frey filament before a tourniquet was applied. In these mice, the tail-flick response to the touch of the von Frey filament after the application of a tourniquet would not necessarily be due to the presence of post-ischaemic allodynia. Therefore, all mice were first screened by testing their tail-flick response to a piece of von Frey hair. The mice that did not flick their tails would proceed onto the next step of the screening procedure, the pinprick test. This test was performed in order to determine if a mouse was capable of responding to a normally painful stimulus by withdrawing its tail. The mice that responded positively to the pinprick by withdrawing their tails were recruited in the experiment. Since the skin thickness, sensitivity and tolerance level to painful stimuli among different mice might vary, these two tests ensured that the mice recruited in the experiment were of comparable sensitivity to touch and pinprick pain at their tails. 25 2.2.2. The Device for Inducing Allodynia in a Mouse Tail: The Tourniquet A tourniquet, as described in section 1.3.2, is an instrument that compresses blood vessels when applied around an extremity to control the circulation and prevent the flow of blood to or from the distal area. The tourniquet used in the experiment was made of a piece of 3 mm long plastic C-Flex tubing (Cole-Parmer Instrument Company, Vernon Hills, Illinois) (Figure 1). The specifications of the C-Flex tubing are as follows: Outer diameter 1/8 inch; inner diameter 1/16 inch; wall thickness 1/32 inch. A special device (see Figure 1) was made for cutting the tubing. This devise had 4 pieces of 3mm thick acrylic glass mounted on a stand. The gaps between these 4 pieces of acrylic glasses were just wide enough for inserting a surgical blade into it. Holes (diameter = 1/8 inch) that were drilled through the four piece of acrylic glass allowed the insertion of the C-Flex plastic tubing. In order to cut the C-Flex plastic tubing into a consistant length of 3mm, the tubing was first inserted through one of the holes until it just reached the other end of the device. The surgical blade was than inserted through the gaps betweenjhe acrylic glasses. By this method, C-Flex tubing tourniquets of consistent lengths of 3mm were obtained. The ischaemic condition in a mouse tail was induced by the application of a tourniquet around the base of the tail (at a site where the tail-diameter was 3.5 mm) (Figure 2). 2.2.3. The Apparatus for Inducing Pain in Mice: Pinprick Device The mouse tail-withdrawal response to a normally painful stimulus was assessed using a 15-gram pinprick device (Figure 1). It was made up of two syringes (a 5-cc and a 10-cc syringes). A 19-14 G needle, which was blunted by filing, was attached to the 26 inner 5-cc syringe. The needle was blunted in order to avoid undesirable injury to the skin of the mouse. Metal ball bearings were put inside this syringe in order to increase its weight to 15 grams. It was then inserted into a 10cc syringe (piston removed). This allowed the inner 5-cc syringe to move freely inside the outer 10-cc syringe. This apparatus was lowered very slowly, which minimized acceleration that could affect the force acting on the skin at the tip of the needle, and vertically above the tail by holding the outer syringe during the pinprick test. If the pinprick device was not applied vertically above the tail, the resultant force induced by the apparatus would be less than 15 grams. The magnitude of the force induced by the apparatus could be calculated from the angle between the vertical plane above the tail and the plane of the pinprick device. The larger the angle, the weaker was the induced force on the skin. Therefore, the apparatus was applied vertically above the tail, so that the inner syringe would exert a constant weight of 15 grams onto the mouse's tail every time when the pinprick test was performed. Figure 3 illustrated the 4 positions on a mouse tail that were tested. Pinprick devices of 5g and 10g had been tested, but it was found that these devices were not heavy enough to induce the tail-flick withdrawal responses in all the mice tested. However, it was found that a pinprick of 20g or higher could scratch the skin of the mouse tails. Therefore, a pinprick device of 15g was chosen in our experiments. 2.2.4. The Apparatus for Assessing Allodynia in Mice: The Von Frey Filaments Four pieces of filaments that could induced forces of 3.9 ± 0 . 1 N, 37.8 ± 0.5 N, 84 ± 1 N, and 178 ± 1 N over areas of 2.0 x 10"7 m 2 , 4.1 x 10"7 m 2 , 6.6 x 10"7 m 2 , and 27 9.5 x 10"7 m 2 , respectively, were used (Figure 1). They were labelled as V F H #1, #2, #3 and #4 according to their stiffness, where V F H #1 was the lightest V F H and V F H #4 was the stiffest V F H . The filaments were pushed towards a mouse tail at various positions (illustrated in Figure 4) until it was slightly bent. However, the response-patterns of an allodynic mouse to all 4 filaments were generally the same. Therefore, unless otherwise specified, only the results obtained by the one that could induce a force of 37.8 ± 0.5 N (VFH # 2) will be discussed in detail. 2.2.5. The Device for Assessing Motor Function of Mice: The Rotarod According to Cartmell et al. (1991), "tests of putative antinociceptive agents that rely on a motor response of an experimental animal to a noxious stimulus will give false positive results, and may be unethical, if the agent compromises motor function". Therefore, the motor function of the mice was assessed with a rotarod. The mice were placed on the rotarod during the experiment. The duration that they could walk on the rotarod was measured. The time from the onset of running until the mouse fell off the rod served as each mouse's performance time. The circumference of the rotarod was 9.9 cm and the rotating speed of the rod was 20 r.p.m. The maximum running time was limited to 10 seconds. If a mouse could walk on the rotarod for 10 seconds, no impairment in its motor function was assumed. 28 Figure 1: The apparatus used in the experiment, (a) Pinprick device; (b) Von Frey hairs; (c) C-Flex plastic tubing; (d) a device for cutting the C-Flex tubing with pre-defined widths (3 mm) between the gaps for blade insertion; (e) 3 mm long C-Flex tubing (Tourniquet). 29 Figure 2: A mouse with a tourniquet applied around the base of its tail. The tourniquet (indicated with an arrow in the picture) was applied at a site where the tail diameter was 3.5 mm. 30 Figure 3: Pinprick test. The pinprick device was lowered slowly and vertically above the mouse tail. There were 4 positions tested, indicated with white arrows. The tourniquet (indicated with an arrow) was applied at a site where the tail diameter was 3.5 mm. Only the response to pinprick at Position P, which was proximal to the tourniquet-site, and at position D, which was distal to the tourniquet-site, are discussed in detail in the result section. 31 Figure 4: Von Frey hair test. The von Frey hair was pushed towards the tail of the mouse until it was slightly bent. There were 4 positions tested, indicated with white arrows. The tourniquet (indicated with an arrow in the picture) was applied at a site where the tail diameter was 3.5 mm. Only the responses to VFH at Position P, which was proximal to the tourniquet-site, and at position D, which was distal to the tourniquet-site, are discussed in detail in the result section. 32 2.2.6. Standard Analgesic Agents Tested The following is a list of analgesic, anaesthetic and control drugs tested in the experiments: morphine (Sigma, St. Lewis, MO, USA), A S A (Sigma, St. Lewis, MO, USA), lidocaine (Sigma, St. Lewis, MO, USA), bupivacaine (Sigma, St. Lewis, MO, USA), QX-314 (Alomone Labs, Jerusalem, Israel), QX-222 (Alomone Labs, Jerusalem, Israel), rocuronium (Sigma, St. Lewis, MO, USA), and pentobarbital (Sigma, St. Lewis, MO, USA). The inhalation general anaesthetic used in the experiments was isoflurane (Abbott Laboratories, Montreal, Canada). All the above analgesic, anaesthetic and control drugs (except isoflurane) tested in the experiments were dissolved in or diluted with normal physiological saline. The pH of the A S A solution was adjusted to 7.4 with sodium hydroxide (NaOH) (1M) in order to minimize the effects of acidic pH of the injected solution on the mice. 2.3. Methods The study of tourniquet-induced post-ischaemic allodynia was divided into four phases. Phase I analysed the time course of tourniquet-induced post-ischaemic allodynia. This phase included the investigation of the relationship between the duration of tourniquet application and the characteristics of the induced allodynia in terms of its onset and duration. A "desirable duration of tourniquet application" was determined from this experiment for the investigation of the mechanism of allodynia development. A desirable duration of tourniquet application was defined as the duration of tourniquet application that could induce post-ischaemic allodynia of sufficient duration for the testing of analgesic drugs. The duration of ischaemia that could induce ischaemic nerve 33 block in a mouse tail was also determined in this phase of the study. In phase II, III and IV of our study, we investigated the pharmacology associated with tourniquet-induced post-ischaemic allodynia in mice. We demonstrated how the method we used to induce allodynia in mice could be applied on the study of the mechanism of and treatment for tourniquet-induced post-ischaemic allodynia. Phase II of the study determined the doses of the analgesic drugs that were effective in blocking pinprick pain at a mouse tail. These drugs (of doses determined in Phase II) were also tested in the Phase III of the study, in which the effects of these analgesic drugs on tourniquet-induced post-ischaemic allodynia was investigated. Various routes of administration of these drugs were tested in Phase II and III of the study in order to investigate their effects on the mice's perception of pinprick pain and post-ischaemic allodynia. Phase IV of the experiment investigated the effects of the analgesic drugs tested in previous phases of the study on the development of tourniquet-induced post-ischaemic allodynia. Drugs that were capable of blocking pinprick pain and/or post-ischaemic allodynia were tested in this phase of the study. The experimental procedures were approved by the Committee on Animal Care, University of British Columbia, Vancouver, British Columbia, Canada. 34 2.3.1. Phase I: Investigation of Tourniquet Induced Post-ischaemic Allodynia 2.3.1.1. The Relationship between the Duration of Tourniquet Application and the Induced Post-ischaemic Allodynia In this set of experiments, we attempted to quantify tourniquet-induced post-ischaemic allodynia with respect to its time course, which included the onset and total duration of the appearance of the induced allodynia. Previous experiments performed in our laboratories showed that the application of a 60-minute tourniquet around the base of a mouse tail could induce allodynia in the tail for duration of 5 - 6 hours. Durations of tourniquet tested in this experiment were 10 minutes, 20 minutes, 30 minutes, and 60 minutes. There was also a control group of mice where no tourniquet was applied. After the initial screening procedures, one of the duration of tourniquet application listed in the previous paragraph was randomly assigned to each recruited mouse. After the randomisation procedures, either a tourniquet with an opaque cover or only an opaque cover (control group) was applied around the base of the mouse tail. The tourniquet was applied at a site around the base of the mouse tail where the tail-diameter was 3.5 mm. V F H test was immediately performed after the tourniquet was released. All the mice were subjected to a follow-up period of 6 days, during which time, the V F H test was performed at 24 hours intervals. 35 2.3.1.2.Effects of Ischaemia on Pinprick Pain This set of the experiments was designed to determine the maximum duration of tourniquet that could be applied around a mouse tail without inducing ischaemic nerve blocks in the tail. The pinprick test was performed at various positions on a mouse tail during the tourniquet period at 2-minutes interval. This experiment was performed together with the previous set of experiments, where the pinprick test was performed during the tourniquet periods. 2.3.2. Phase II: Effects of Standard Analgesic Agents on Pinprick Pain in Normal Mice 2.3.2.1. The Effects of Systemic Administration of the Analgesic Drugs on Pinprick Pain in Normal Mice A dose of test-drug or saline control of volume 0.5 ml was randomly assigned to and injected into the mouse either subcutaneously at the back of the neck (sc) or intraperitoneally (ip). The experimenter was blinded, such that he/she did not know the identities of the injected drugs. The pinprick test was then performed immediately following the injection. If the injected drug had no effect on the tail-flick responses of the mice to pinprick, the experiment was discontinued at 90 minutes after the injection of the test-drugs or saline control. Otherwise, the pinprick test was continued until the pinprick responses of the mice were observed again. In experiments performed earlier in our laboratory, the analgesic agents were injected subcutaneously at the back of a mouse neck. The averaged onset time for most of the tested analgesics was about 10 minutes. The route of administration was 36 changed in recent experiments because a faster onset of action was needed. Intraperitoneal injection of the same analgesic drugs at the same doses was found to be effective in reducing the onset time from about 10 minutes to approximately 3-6 minutes. This reduction in onset time was favourable for the experiment that is described in the next section. All the drugs tested were dissolved in normal physiological saline. The volume of injection was 0.5 ml. The drugs tested were: morphine (5 mg/kg, 10 mg/kg, 20 mg/kg), lidocaine (40 mg/kg, 75 mg/kg), bupivacaine (50 mg/kg), QX-222 (140 mg/kg), pentobarbital (30 mg/kg, 40 mg/kg), rocuronium (0.7 mg/kg) and saline control (0.5 ml). 2.3.2.2.The Effects of Systemic Administration (with the Tail Occluded by a Tourniquet) of the Analgesic Drugs on Pinprick Pain in Normal Mice This set of experiments was designed to determine if a drug that relieved the pinprick pain in the previous set of experiments possessed a site of action in the C N S . A tourniquet was employed to occlude the blood flow to the tail, and, hence, inhibit the distribution of the drug to the parts of the mouse that were distal to the tourniquet-site. In this case, a drug that blocked the pinprick response would presumptively be due to central analgesic actions. However, prolonged duration of ischaemia could induce ischaemic nerve block in the peripheral nerves, resulting in the absence of the pinprick response. The actual analgesic properties of the injected drugs could have been masked by the ischaemia-induced blockade of the pain fibres. Therefore, the tourniquet (for blood occlusion) was applied for no longer than 20 minutes, which was determined 37 from the experiments described in section 2.3.1.2. The drug was injected intra-peritoneal^ immediately after the tourniquet was applied. The volume of injection was 0.5 ml and the drug tested was lidocaine (75 mg/kg). 2.3.2.3. The Effects of Peripheral Administration of the Analgesic Drugs on Pinprick Pain in Normal Mice Among the analgesic agents described and used in pervious experiments, some might induce analgesia when administered peripherally. The drugs tested were morphine (1.5%), lidocaine (1%), bupivacaine (0.3%), QX-314 (2%), A S A (2.4%) and saline control. All the drugs tested were dissolved in a saline solution. The volume of injection was 0.05 ml. These drugs were tested again in this set of experiments in order to determine their effects on pinprick response when they were injected intravenously into a mouse tail (circulation to/from the tail was stopped by the application of a tourniquet around the base of the tail). After the screening procedure, a mouse was anaesthetized with isoflurane (2%) by placing the mouse into a 5L anaesthetic chamber, which was filled with 2% isoflurane. The double cuff method was used in this experiment. A 400mmHg pressure cuff was applied at the base of the mouse tail as soon as the righting and withdrawal reflexes of the mouse were lost. A dose of the analgesic drugs or control drugs was randomly assigned to the mouse and was injected intravenously (iv) into a site that was close to the tip of the tail. A tourniquet (C-Flex tubing) was then applied around the tail (at a tail diameter of 3.5 mm). The 400mmHg pressure cuff was released and the 38 mouse was immediately removed from the anaesthetic chamber. The pinprick test was performed as soon as the mouse was removed from the anaesthetic chamber and the pinprick response of the mouse was recorded. If the pinprick response was absent after the injection, pinprick test was continued to be performed until the mouse respond to pinprick positively again. Otherwise, the experiment was discontinued at 90 minutes after the drugs/saline control was injected. 2.3.3. Phase III: Effects of Standard Analgesic Agents on Tourniquet-Induced Post-ischaemic Allodynia in Mice 2.3.3.1. The Effects of Systemic Administration of Analgesic Agents on Tourniquet-Induced Post-ischaemic Allodynia in Mice In this experiment, the effects of analgesic agents on the tail-flick responses of the allodynic mice to touch were evaluated. Post-ischaemic allodynia was induced by the application of a 30-minute or a 60-minute tourniquet around the base of a mouse tail. After the required tourniquet-period, the presence of allodynia in the mouse tail was tested with a piece of V F H . If a mouse was found to be allodynic for 30 minutes after the release of the tourniquet, one of the test drugs or saline control was injected either subcutaneously (sc) at the back of the neck or intraperitoneally (ip). The drugs and saline were randomly assigned to the allodynic mice. The presence of allodynia in the mouse tails was continued to be tested with the von Frey filament. The ability of the mice to walk on a rotarod was also tested. If an allodynic mouse stopped responding to touch by flicking its tail, the V F H test was continued up to several hours or until the 39 mouse again flicked its tail upon the touch. The presence of allodynia in a mouse tail was tested again on the next day if the tail-flick response to touch was blocked completely on the first day of the experiment. Otherwise, the experiment was discontinued at 90 minutes after the drugs/saline was injected. The following drugs were tested: Morphine (5, 10, and 20 mg/kg), lidocaine (75 mg/kg), QX-222 (140 mg/kg), bupivacaine (40, 50 mg/kg), pentobarbital (30, 40 mg/kg), rocuronium (0.7 mg/kg), and saline as control (0.5 ml). All the drugs tested were dissolved in a saline solution. The volume of injection was 0.5 ml. 2.3.3.2.The Effects of Systemic Administration (with the Tail Occluded by a Tourniquet) of Analgesic Agents on Tourniquet-Induced Post-ischaemic Allodynia in Mice This experiment was aimed at determining if the analgesic drugs that blocked the expression of tourniquet-induced post-ischaemic allodynia in the previous experiment acted through a central mechanism. The experimental procedures were very similar to that mentioned in section 2.3.2. After initial screening procedures described in section 2.2.1, post-ischaemic allodynia in mice was induced by a 30-minute tourniquet. The mice that responded positively to the von Frey filament for at least 30 minutes after the release of the tourniquet were anaesthetized with 2% isoflurane in a 5L Plexiglas chamber, during which time, a tourniquet was applied again around the tail at the same position as the first tourniquet. The purpose of anaesthetizing the mice was to minimize the pain experienced by the mice when the second tourniquet was being applied around their allodynic tails. As soon as the tourniquet was applied, the mouse was 40 removed from the anaesthetic chamber followed by intraperitoneal injection of one of the following drugs: lidocaine (75 mg/kg), morphine (10 mg/kg, 20 mg/kg) or saline (control). All the drugs tested were dissolved in a saline solution. The volume of injection was 0.5 ml. The drugs and saline control were randomly assigned to the allodynic mice. The duration of the second tourniquet was 20 minutes, during which time V F H test was performed at all positions mentioned. 2.3.3.3.The Effects of Peripheral Administration of Analgesic Agents on Tourniquet-Induced Post-lschaemic Allodynia in Mice The effects of the analgesic drugs on the expression of tourniquet-induced post-ischaemic allodynia peripherally were tested in this set of experiments. Post-ischaemic allodynia in a mouse tail was induced by a 30-minute tourniquet. A mouse that was allodynic in the tail for at least 30 minutes after the tourniquet was released was anaesthetized with 2% isoflurane. A 400 mmHg pressure cuff was applied around the base of the mouse tail in order to occlude blood flow to the tail. One of the test drugs was immediately injected intravenously at a site that was close to the tip of the tail (distal to the pressure cuff). This was followed by the application of a second tourniquet (C-Flex tubing), which trapped the drugs in the tail, at a position where the tail diameter was 3.5 mm. Then, the 400 mmHg pressure cuff was released and the mouse was removed from the anaesthetic chamber. The presence of allodynia at various positions of the mouse tail was then tested with a piece of V F H . All the test drugs were dissolved in a saline solution. The volume of injection was 41 0.05 ml. The following is a list of drugs that were tested and randomly assigned to the mice in this set of experiments: morphine (1.5%), lidocaine (1%), bupivacaine (0.3%), QX-314 (2%), and saline as control (0.05 ml). 2.3.4. Phase IV: Effects of Standard Analgesic Agents on the Development of Tourniquet-Induced Post-lschaemic Allodynia in Mice 2.3.4.1. The Effects of Systemic Administration of Analgesic Drugs on the Development of Tourniquet-Induced Post-lschaemic Allodynia in Mice In this set of experiments, we investigated the effects of various analgesic agents on the development of tourniquet-induced post-ischaemic allodynia in a mouse tail when injected systemically prior to the application of a tourniquet. The drugs tested were: lidocaine (75 mg/kg), morphine (20 mg/kg), A S A (100 mg/kg, 300 mg/kg, 400 mg/kg), and saline (control). All drugs were dissolved in saline and the volume injected was 0.5 ml. The duration of tourniquet application used in this set of experiments was 20 minutes. The V F H test was performed immediately after a tourniquet was applied. It was continued to be performed after the tourniquet was released. All mice were subjected to a 6-day follow-up period, during which time the V F H test was performed at 24-hour intervals. 42 2.3.4.2. The Effects of Inhalation General Anaesthetics on the Development of Tourniquet-Induced Post-ischaemic Allodynia in Mice This experiment was aimed at determining if the development of tourniquet-induced post-ischaemic allodynia in a mouse tail requires conscious perception of pain. After initial sensitivity screening procedures described in section 2.2.1, all recruited mice were anaesthetized by 2% isoflurane in a 5L Plexiglas chamber. A tourniquet was applied around the base of a mouse tail after it had been anaesthetized for 30 minutes. The mouse was kept anaesthetized during the 30-minute tourniquet period as well as the first 30 minutes after the tourniquet was released. There was also a control group of mice which were also anaesthetized in the same way; however, no tourniquet was applied around their tails. The mice were randomised such that they had equal chances of participating in the tourniquet group or the control group. The V F H test was performed immediately after the mice were removed from the anaesthetic chamber for the next 280 minutes. All mice were subjected to a 6-day follow-up period, during which time the V F H test was performed at 24-hour intervals. 2.3.4.3. The Effects of Peripheral Administration of Analgesic Drugs on the Development of Post-ischaemic Allodynia in Mice This set of experiments determined the role of peripheral nerves in the development of tourniquet-induced post-ischaemic allodynia. After initial sensitivity screening (section 2.2.1), all mice were anaesthetized by 2% isoflurane in a 5L Plexiglas chamber. A pressure cuff (400 mmHg) was applied at the base of a mouse 43 tail to occlude circulation toward the tail. The drugs were randomly assigned to the mice. One of the analgesic drugs or saline control of volume 0.05 ml was injected intravenously into the tail. This was followed by the application of a tourniquet at a position where the tail-diameter was 3.5 mm. The duration of tourniquet employed in this set of experiments was 20 minutes. The pressure cuff was released immediately after the tourniquet was applied, and the mouse was removed from the anaesthetic chamber. The V F H test was performed immediately after the mouse was removed from the anaesthetic chamber. The tourniquet was released after it had been applied for 20 minutes. If the mouse responded negatively to the V F H test, the pinprick test would be performed at position D. All mice were subjected to a 6-day follow-up period, during which time V F H test was performed at 24-hour intervals. The following is a list of drugs that were tested and randomly assigned to the mice in this set of experiments: Morphine (1.5%), lidocaine (1%), bupivacaine (0.3%), QX-314 (2%), and saline control (0.05 ml). All the test drugs were dissolved in a saline solution. The volume of injection was 0.05 ml. There was also an experiment in which lidocaine (2%) was injected subcutaneously at a site that was proximal to the tourniquet-site in order to block the pain fibres innervating the mouse tail. The nerve block induced by lidocaine was confirmed by performing pinprick test on the mouse tail at positions that were distal to the injection site. A tourniquet was applied around a mouse tail (at tail diameter of 3.5 mm) only if the injected lidocaine successfully induced a blockade of the pinprick response of the mouse. The duration of the tourniquet was 15 minutes. The volume of injection was 0.03 ml. V F H test was performed immediately after the tourniquet was 44 released. All mice were subjected to a 6-day follow-up period, during which time V F H test was performed at 24-hour intervals. 2.3.5. Data Analysis for Phase II, III and IV of the Study Statistical evaluation of the results was performed using Chi-square test. The proportion of the mice responded to pinprick/touch of each analgesic treated group was compared with that of saline control group. The null hypothesis was: in the sampled population, the proportion of the mice that responded to pinprick/touch of the drug treated group was the same as that of the (saline) control group. P<0.05 was considered significant. 45 3. RESULTS 3.1. Phase I: Investigation of Tourniquet-Induced Post-lschaemic Allodynia in Mice 3.1.1. Time Course of Post-lschaemic Allodynia As described in section 2.3.1.1, in this set of experiments, we investigated the relationship between the duration of tourniquet and the induced allodynia in a mouse tail. Post-ischaemic allodynia in a mouse tail was assessed by a piece of V F H . By the experimental definition of allodynia (section 2.1), a flick of the tail upon the touch of the filament indicated the presence of allodynia in a mouse tail. In summary, the following two characteristics of post-ischaemic allodynia were observed with an increasing duration of tourniquet application: 1) Increase in latency of the appearance of tourniquet-induced post-ischaemic allodynia 2) Increase in the total duration of the tourniquet-induced post-ischaemic allodynia The tip of a mouse tail was found to be more sensitive to pinprick and touch than other positions of the tail. However, the patterns of tail-flick response to pinprick at positions that were distal to the tourniquet-site were very similar, despite the difference in sensitivity among these positions. The response pattern of a mouse to touch at position P (proximal to the tourniquet) was found different from that at other positions that was distal to the tourniquet-site. Therefore, the responses of the allodynic mice to touch at positions that were proximal (position P) and distal (position D) (see Figure 4) 46 to the tourniquet-site were discussed in detail. 3.1.1.1.Latency of the Appearance of Post-lschaemic Allodynia The 'latency of the appearance of post-ischaemic allodynia' was defined as the duration from the release of the tourniquet to the time at which allodynia was first detected in the mouse tail (at position D). At position D, the latency of the appearance of allodynia increased with increasing duration of tourniquet application. By linear correlation analysis, the relationship between the duration of ischaemia and the latency of the appearance of allodynia was found to be statistically insignificant (P=0.062); but the relationship between the log value of the duration of ischaemia and the log value of the latency of the appearance of allodynia (in those mice that developed allodynia) was found to be statistically significant (P=0.032), with a correlation coefficient of 0.97 (Figure 5). Slope of the linear regression line (shown in Figure 5) was 0.8±0.3, and the y-intercept was -0.4±0.5. Logarithmic transformation of the data was performed because the linear correlation coefficient (0.97) calculated in this way was higher than that calculated from the original data (not transformed). The delay in the appearance of allodynia at position D in the groups that received long durations of tourniquet application might have resulted from a nerve block induced by the prolonged ischaemia in the tails (see section 3.1.2). 47 The relationship between Log(latency of the appearance of allodynia) and Log (duration of ischaemia) o . o - l 1 1 1 1 — i 1 0.50 0 .75 1.00 1.25 1.50 1.75 2 .00 Log (duration of ischaemia) Figure 5: The relationship between the log (latency of appearance of post-ischaemic allodynia) and log (duration of ischaemia). Only the latency of appearance of allodynia at position D of the mouse tail was shown in this graph. 48 3.1.1.2.Duration of Post-ischaemic Allodynia At both positions D and P, the duration of post-ischaemic allodynia was found to be dependent on the duration of tourniquet application. Notably, in the control group, none of the mice responded positively to the touch of a piece of V F H at any tested position in the first 300 minutes of the experiment as well as in the 6-day follow-up period. In the figures, t = 0 minute was defined as the time at which the tourniquet was released. In the group that received a 10-minute tourniquet (n=5), only 1 out of the 5 mice in this group was found to be allodynic at both positions D and P. This mouse was found to be allodynic at both positions throughout the 6-day follow-up period. (Figure 6a, Figure 7a) In the group that received a 20-minute tourniquet (n=5), 100% of the mice developed allodynia at position D, but only 40% of them developed allodynia at position P. At position D, 100% of the mice were allodynic until 50 minutes after the tourniquets were released. A total of 3 (out of 5) mice in this group recovered completely from the induced allodynia on the first day of the experiment. The percent of mice that were allodynic at position D dropped to 60% and 40% at t = 120 minutes and t = 300 minutes after the tourniquet was released respectively. At the end of the 6-day follow-up period, 80% of the mice were found completely recovered from the induced allodynia at position D; moreover, none of them was found to be allodynic at position P starting on the 3 r d day of the experiment (Figure 6b, Figure 7b). In the group that received a 30-minute tourniquet (n=5), 100% of the mice developed allodynia at position D, and 80% of them developed allodynia at position P. 49 None of the mice in this group recovered from the induced allodynia at either position D or P on the first day of the experiment. The percent of mice that were allodynic at position D dropped to 80% and 40% on the 3 r d and 4 t h day of the experiment respectively. On the 6 t h day of the experiment, 40% of the mice in this group were still allodynic at position D and 60% of them were still allodynic at position P. (Figure 6c, Figure 7c) In the group that received a 60-minute tourniquet (n=5), 100% of the mice developed allodynia at position D, but only 60% of them developed allodynia at position P. 1 out of the 5 mice recovered from allodynia at position D within the first 300 minutes after the tourniquet was released. None of the other 4 mice recovered from the tourniquet-induced post-ischaemic allodynia at position D in the 6-day follow-up period. 60% of mice in this group were found to be allodynic at position P at t = 300 minutes, but the allodynia developed very slowly in these mice at this position. (Figure 6d, Figure 7d) Normally, once a mouse recovered from the induced allodynia, it remained recovered (not responding to touch) throughout the rest of the 6-day follow-up period. However, as seen in the figures listed in the following pages, occasionally, some recovered mice were found to be allodynic again. The lack of responses to the touch of a V F H was often observed when the allodynic mice were moving constantly in their mouse holders. Their movements could have distracted them from the touch of a V F H , resulting in false negative responses to the touch. Therefore, when interpreting the results, the focus should be placed on the general trends of the responses of the mice to the touch. 50 Positions D and P 10-Minute Tourniquet 100-1 tt I 8 0 -UL > o Time (min) —— Position D --o-- Position P (a) Figure 6: Time-course of tourniquet-induced post-ischaemic allodynia at positions D and P (first 300 minutes after the tourniquet was released). Sample size in each group of mice = 5. The tourniquets were released at t = 0 minute. Each point indicated the actual percent of mice that were allodynic at position D or P at the corresponding time. The durations of tourniquet application tested were 10 minutes, 20 minutes, 30 minutes, 60 minutes, and the results were shown in graphs (a), (b), (c) and (d), respectively. 51 Positions D and P 20-Minute Tourniquet —— Position D --o-- Position P (b) 52 Positions D and P 30-Minute Tourniquet 2 0 0 3 0 0 Time (min) —— Position D --o-- Position P (c) 53 Positions D and P 60-Minute Tourniquet 100 - , CM I 80 > O •4-" .E 6 0 H c o Q. w 4 0 - j 0^  o 20 «3ES^SX>0006 100 2 0 0 Time (min) —— Position D -o - Position P '0 3 0 0 (d) 54 Positions D and P 10-Minute Tourniquet 100-1 CM tt I 80-> O Time (hr) ----- Position D --o-- Position P (a) Figure 7: Time-course of tourniquet-induced post-ischaemic allodynia at positions D and P (6-day follow-up period). Sample size in each group of mice = 5. The tourniquets were released at t = 0 minute. Each point indicated the actual percent of mice that were allodynic at position D or P at the corresponding time. The durations of tourniquet application tested were 10 minutes, 20 minutes, 30 minutes, 60 minutes, and the results were shown in graphs (a), (b), (c) and (d), respectively. 55 Positions D and P 20-Minute Tourniquet lOOm CM X 8 0 -> o c 6 0 -G) C T3 c o Q. in 0) i _ 0) u 4 0 - t o * 2 0 -o4- —I— 20 O i 100 —$ 120 4 0 60 80 Time (hr) —— Position D --o-- Position P (b) 56 Positions D and P 30-Minute Tourniquet Positions D and P 60-Minute Tourniquet —— Position D o- Position P (d) 58 3.1.1.3.Recovery from Tourniquet-Induced Post-ischaemic Allodynia In general, during the first few hours after tourniquet release, allodynic mice responded to the touch of the von Frey hairs by withdrawing their tails vigorously. On the next day of the experiment, some of the mice stopped responding to the touch while others responded to the touch less vigorously. The tail-flick responses to the touch of the V F H #2, which exerted a force of 37.8 + 0.5 N, has been discussed previously. For determining whether the mice recovered from the induced allodynia gradually or suddenly, the responses of the mice to the touch of the other 3 von Frey hairs are relevant and presented in this section. A scoring system was used in describing the responses of the mice to the touch of different von Frey hairs. It was found that if a mouse responded to the touch of the lightest V F H , it would also respond to the touch of other stiffer filaments. If a mouse responded to the touch of all 4 von Frey hairs, a von Frey hair score (VFH score) of 4 was given to the mouse at the corresponding time in the experiment. If a mouse did not respond to the touch of the least stiff V F H (i.e. V F H # 1, see section 2.2.4) by flicking its tail but to the others, a score of 3 was assigned to the mouse, and so on. A mouse that did not respond positively to the touch of any of the 4 von Frey hairs would receive a score of 0. As seen in Figure 8, in the first 15 minutes after the tourniquet-release, the averaged V F H score of each group of mice increased rapidly. As time went by, the mice began to stop responding to lighter VFH(s), resulting in the decrease in their averaged V F H scores. It was found that the mice recovered gradually, rather than abruptly, from the induced allodynia in their tails. This was indicated by the gradual decrease in their V F H scores in increments of less than multiples of 0.8 over the 6-day experimental 59 period. If the mice stopped responding to the touch of all 4 V F H suddenly, since there were 5 mice in each group, the averaged V F H score of this group of mice would have decreased in multiples of 0.8. However, in the experiment, each decrease in the averaged V F H score was less than 0.8; therefore, we concluded that the mice recovered gradually from the induced allodynia in their tails. The recovery rate of the tourniquet-induce post-ischaemic allodynia was defined as the rate of which each group of allodynic mice recovered from the induced allodynia. The recovery rates of allodynia of each group of mice were obtained and compared by performing linear regression analysis on the data. Only the groups in which 100% of the mice were found to be allodynic after the release of their tourniquets were analysed. In the 6-day follow-up period (Figure 9), the slopes of the regression lines for the groups of mice that received 20-minute, 30-minute and 60-minute tourniquets were found to be -0.44±0.14, -0.49±0.10, and -0.09±0.11 % of mice/min, respectively. That is, late recovery was at roughly the same rate in the 20- and 30-minute tourniquet group. In the 60-minute tourniquet group 4 of 5 mice did not recover significantly in the 5 days after the experiment. 60 Test for Allodynia at Position D •w 1 1 1 1 1 1 0 50 100 150 2 0 0 2 5 0 3 0 0 Time (min) 10 Min. Tourniquet A 20 Min. Tourniquet - v - 30 Min. Tourniquet — o — 6 0 Min. Tourniquet (a) Figure 8 The magnitude of tourniquet-induced post-ischaemic allodynia in mice. Each point on the graphs represents the averaged von Fray hair score of each group of mice (n=5) at the corresponding time. Graphs (a) and (b) show the responses of the mice to the touch of the VFHs at position D and P, respectively, in the first 300 minutes of the experiment. Graphs (c) and (d) show their responses to the touch of the VFHs at position D and P, respectively, in the 6-day follow-up period. 61 Test for Allodynia at Position P • + 1 1 1 1 1 1 0 50 100 150 2 0 0 2 5 0 3 0 0 Time (min) -•»-• 10 Min. Tourniquet 20 Min. Tourniquet 30 Min. Tourniquet —0—60 Min. Tourniquet (b) Test for Allodynia at Position D s N V . * k -A- - . . ^ V - . •i —I 1 I 1 1 1 2 0 4 0 6 0 80 100 120 Time (hours) 10 Min. Tourniquet 20 Min. Tourniquet -v - 30 Min. Tourniquet - o - 6 0 Min. Tourniquet (c) - » - • 10 Min. Tourniquet - -A - - 20 Min. Tourniquet - v - 30 Min. Tourniquet —o— 60 Min. Tourniquet (d) Rate of recovery from allodynia at position D in 6-days follow-up period (n = 5) 100-fiO c '•5 c O CM W * l i -CU > o o 80-1 60-4 0 20-0- —i— 20 4 0 —i— 60 80 100 120 Time (hr) • 20 Min. Tourniquet o 30 Min. Tourniquet • 60 Min. Tourniquet — 20 Min. Tourniquet 30 Min. Tourniquet 60 Min. Tourniquet Figure 9 The recovery rates of tourniquet-induced post-ischaemic allodynia in mice. Each point on the graphs represent the percent of mice in each group (n=5) responded to the touch of a piece of VFH. The lines are the best-fit regression lines for each group of mice. The above graph shows the responses of the mice to touch in the first 300 minutes and in the 6-day follow-up period respectively. 65 3.1.2. Effects of Ischaemia on Pinprick Pain In this experiment, the pinprick test was performed at various positions on the mouse tails during various tourniquet-periods. It was found that the mice began to stop responding to pinprick at position D at about 15 minutes after the tourniquets were applied. In the groups that received 30-minute and 60-minute tourniquets, all of the mice had stopped flicking their tails upon pinprick at 26 minutes and 34 minutes after the tourniquets were applied, respectively. (Figure 10) Position P was the only tested position that was proximal to the tourniquet-site. Although some mice had stopped responding to pinprick at position D during the tourniquet-periods, 100% of the tested mice responded positively to pinprick at position P during the entire tourniquet-periods. Therefore, in the experiments that required the application of a second tourniquet to occlude the blood flow to the tail, (see below) the duration of the second tourniquet was limited to 20 minutes. Otherwise, the actual analgesic action of an injected drug might have been masked by the ischaemic nerve block induced by the application of a tourniquet. These results suggest that the delay of appearance of post-ischaemic allodynia after 30-/60-minute tourniquet application (see above) may have been related to blockade of nerve impulse initiation/conduction during the application of the tourniquet. 66 Pinprick Test at Position D (20-minute tourniquet) c •E c o • Q., W 0) o " u 100-90COOOOO 80 £ 60-Q. C 5. 4 0 20 — i 1 1 1 1 1 0 10 2 0 30 4 0 50 60 Time (min.) (a) Figure 10: The effect of ischaemia on pinprick response at position D. The graphs only show the pinprick response of the mice during the tourniquet period. The graphs of durations of tourniquet application of (a) 20 minutes, (b) 30 minutes, and (c) 60 minutes (n=5) are shown. 67 Pinprick Test at Position D (30-minute tourniquet) o o Q. O tn >-c IOO-900COCC0 8 0 -6 0 -4 0 20 A 00 00 — i 1—ee# 1 1 1 0 10 2 0 30 4 0 50 60 Time (min.) (b) Pinprick Test at Position D (60-minute tourniquet) c S c o Q. tn o i-o o 100 -ecococcco 80 A Z 6 0 i Q. C Q- 4 0 H 20H 00 00 00 00 — i 1 1 ooajxxxxxpooooo 0 10 20 30 4 0 50 60 Time (min.) (c) 68 3.2. Phase II: Effects of Standard Analgesic Agents on Pinprick Pain 3.2.1 The Effects of Systemic Administration of Analgesic Drugs on Pinprick Pain in Normal Mice In this set of experiments, the analgesic drugs were injected subcutaneously (sc) at the back of the mouse necks or intraperitoneally (ip) into the mice. The volume of injection was 0.5 ml. Pinprick test was performed at the mouse tails immediately following the injections of the drugs. The drugs tested were: morphine (5 mg/kg, 10 mg/kg, 20 mg/kg), lidocaine (40 mg/kg, 75 mg/kg), QX-222 (140 mg/kg), bupivacaine (50 mg/kg), pentobarbital (40 mg/kg, 50 mg/kg), rocuronium (0.7 mg/kg) and saline as control. If the mice stopped responding to pinprick at their tails after the drugs were injected, the pinprick test was continued until the mouse responded positively to pinprick again. Otherwise, the experiment was discontinued at 90 minutes after the drug was injected. From previous work done in our laboratory, it was found that the onset of actions of the analgesic drugs tested was less than 15 minutes. Therefore, we assumed that 90 minutes would be sufficient for confirming whether the injected drugs were effective in inducing analgesia in the tested mice. Injection of saline (control) had no effect on the pinprick response of any mouse in the control group (n=10). Also, no decline in the performance on a rotarod was observed for any mouse in the control group. In summary, compared to the control group of mice, the injections of morphine (10 mg/kg, 20 mg/kg) (sc), lidocaine (75 mg/kg) (sc, ip), and QX-222 (140 mg/kg) (sc) significantly (P<0.05) affected the pinprick responses. However, the injections of lidocaine (40 mg/kg) (ip), rocuronium (0.7 mg/kg) (ip), and pentobarbital (40 mg/kg) had no effect on the pinprick responses of any mice 69 which received these drugs. 3.2.1.1.Morphine (5 mg/kg, 10 mg/kg, and 20 mg/kg) Subcutaneous injections (at the back of the neck) (sc) of morphine (0.5 ml) into normal mice induced a dose-dependent effect on their tail-flick responses to the pinprick test (Figure 11). In the group of mice that received 5 mg/kg morphine (sc) (n=5), only 20% of the mice in this group stopped responding to pinprick for a brief period. However, for the group of mice that received 10 mg/kg morphine (sc) (n=5), 100% of the mice in this group stopped responding to pinprick (n=5) for approximately 170 minutes after the injection. For the group of mice that received 20 mg/kg morphine (sc) (n=5), the pinprick responses were absent in 100% of the tested mice for approximately 280 minutes after the injection. The absence of pinprick responses raised the question that whether the mice were unable to perceive the pinprick pain or unable to withdraw their tails after the injection of morphine. The abilities of all the tested mice to walk on the bench and the spontaneous tail movements of these mice were not affected at any time during the experiment. Therefore, the absence of their pinprick responses after the injection of morphine can be attributed to the analgesia induced by morphine. 70 Pinprick at Position D 0-| I'AIIAIII gmij>oooo6 1 1 1 1 1 0 60 120 180 240 300 360 Time (min) Morphine (5 mg/kg) - - 0 - - Morphine (10 mg/kg) — Morphine (20 mg/kg) Figure 11: Effects of subcutaneous injections of morphine (at the back of the neck) on the pinprick response of mice. Morphine (0.5 ml) of various doses (5 mg/kg, 10 mg/kg, and 20 mg/kg) was injected at t=0 minute (n=5). A dose-dependent blockade of pinprick response was observed. 71 3.2.1.2.Lidocaine (40 mg/kg, 75 mg/kg), QX-222 (140 mg/kg), and Bupivacaine (50 mg/kg) Intraperitoneal (ip) injection of 75 mg/kg, but not 40 mg/kg, lidocaine (0.5 ml) into normal mice significantly (P<0.05) affected their pinprick responses at the tails. For the group of mice that received 75 mg/kg lidocaine (ip) (n=5), the pinprick responses was abolished in 4/5 tested mice for about 40 minutes after the injection (Figure 12). The performance of this group of mice on a rotarod was also impaired for approximately 45 minutes after the drug was injected (Figure 13b). However, their abilities of walking on the bench and their spontaneous tail movements were not affected at any time during the experiment. At about 30-40 minutes after the injections, most of the mice began to regain their abilities to walk on the rotarod, but they were still not responding to the pinprick test at this time of the experiment (Figure 12, Figure 13). Moreover, the mice were able to breathe spontaneously and no ventilation procedures were needed throughout the experimental period. All of these observations suggested that the mice were not paralysed in the experiment, even though their performance on a rotarod was affected by the injected lidocaine. The absence of the tail-flick withdrawal responses to pinprick in these mice can therefore be attributed to an analgesic action of lidocaine. For the group of mice that received 40 mg/kg lidocaine (ip) (n=5), although their pinprick responses were not affected, their performance on the rotarod was slightly affected for about 15 minutes (Figure 13b). For the group of mice that received subcutaneous injection of lidocaine (75 mg/kg) (at the back of the neck) (n=12), 66% of the tested mice had stopped responding to pinprick at their tails (Figure 14a). The time during which no pinprick response was observed in this group of mice was found to be longer than that of the 72 group of mice that received the same dose of lidocaine intraperitoneal^. The pinprick response in this group (sc lidocaine, 75 mg/kg) of mice was suppressed approximately 90 minutes, with an onset time of approximately 10 minutes after the injection. The rotarod performance of this group of mice was also significantly (P<0.05) affected (Figure 14b). However, this group of mice were still able to walk on the bench and their spontaneous tail movements were observed, in spite of the decline of their performance on a rotarod. The average duration on the rotarod of this group of mice dropped to 3 seconds at 10 minutes after the drug was injected. At 30 minutes after the injection, their average duration on the rotarod reached the minimum value of 2 seconds. Then, the mice began to retain their ability to walk on the rotarod. At 110 minutes after the injection, all the mice in this group were able to walk on the rotarod for 10 seconds again. Subcutaneous injection of QX-222 (140 mg/kg) affected the pinprick response in only 30% of the tested mice (n=20) (Figure 15a). Compared to the control group (n=10), the absence of the pinprick response in the group that received QX-222 was found to be statistically significant (P<0.05). The drug was injected subcutaneously at the back of the mouse's neck. The pinprick response was absent for approximately 230 minutes. A higher dose of QX-222 would kill the animal; therefore, it was not tested. The performance of this group of mice on a rotarod was also significantly (P<0.05) affected (Figure 15b). However, the ability of this group of mice to walk on the bench and their spontaneous tail movements were not affected in the experiment. The average duration on a rotarod of this group of mice dropped to about 6 seconds at 40 minutes after QX-222 (140 mg/kg) was injected. Then, the mice began to regain their ability to walk on 73 the rotarod. At about 110 minutes after the injections, all the tested mice were able to walk on the rotarod for 10 seconds again. Subcutaneous injection (at the back of the neck) of bupivacaine (50 mg/kg) had no effect on the pinprick response of any tested mouse (n=10) at any position on the tail. Also, the performance of all the mice tested on a rotarod was not affected after the injections of bupivacaine (50 mg/kg, sc). 74 Pinprick Test at Positions D and P Figure 12: The effects of intraperitoneal injection of lidocaine (75 mg/kg) on the pinprick responses of mice (n = 5). The pinprick responses of the mice at positions D and P are shown. The drug was injected at t = 0 minute. 75 •a o as o OH _ c o o 8 o ro "1 8 7 6 5 -4-3 -2 1 0 Lidocaine (40mg/kg) i —r-5 —i 1 1 1 1 10 15 20 2 5 30 Time (min) (a) 10 -a o L-ro •*-> O Dd _ c 6 o a> o ro v . 3 Q Lidocaine (75 mg/kg) i——i 1 1 1 1 — 0 10 2 0 30 4 0 50 6 0 Time (min.) (b) Figure 13: The effect of intraperitoneal injection of (a) 40 mg/kg and (b) 75 mg/kg lidocaine on the performance of mice on a rotarod (n = 5). The drug was injected at t = 0 minute. Each point on the graphs represents the averaged duration (±SEM) of the mice on a rotarod at the corresponding time. 76 Position D 100 •o O 1 _ CO o 10 8 m 6 O </) 5 2 Q Time (min) Rotarod Performance I" —i— 30 60 —i— 90 120 150 Time (min) 180 (a) 180 (b) Figure 14: The effect of subcutaneous injection (at the back of the neck) of 75 mg/kg lidocaine on normal mice (n = 12). Graph (a) and (b) show the pinprick responses and their performance on a rotarod respectively. The drug was injected at t = 0 minute. For graph (b), each point on the graphs represents the averaged duration (±SEM) of the mice on a rotarod at the corresponding time. 77 Position D 100 0 30 60 90 120 150 180 2 1 0 2 4 0 2 7 0 Time (min) (a) Roratod Performance T3 O i_ co •*-> o 5 ° O (ft CO 3 Q 10 8 6 -4 -—i 1 1 1 1 1 1 1 1 30 6 0 90 120 150 180 2 1 0 2 4 0 2 7 0 Time (min) (b) Figure 15: The effect of subcutaneous injection (at the back of the neck) of QX-222 (140 mg/kg) on normal mice (n = 20). Graph (a) and (b) show the pinprick response of the mice and their performance on a rotarod respectively. The drug was injected at t = 0 minute. For graph (b), each point on the graphs represents the averaged duration (±SEM) of the mice on a rotarod at the corresponding time. 78 3.2.1.3.Pentobarbital (30 mg/kg, 40 mg/kg) Intraperitoneal injection of pentobarbital (30 mg/kg and 40 mg/kg) into normal mice induced a dose-dependent effect on their pinprick responses and a dose-dependent decrease in their performance on a rotarod. For the group of mice that received 30 mg/kg pentobarbital (ip) (n=5), the tail-flick response to pinprick at position D was absent in 20% of the mice for a duration of about 10 minutes after the injection (Figure 16a). The onset time of the effect of pentobarbital (30 mg/kg) on their pinprick responses was found to be 3 minutes. However, the performance on a rotarod of all the mice was also impaired for a period of approximately 30 minutes after the injection of the drug (Figure 16b). The righting reflexes of all of the mice in this group were lost during this period. For the group of mice that received 40 mg/kg pentobarbital (n=5), the tail-flick response to pinprick at position D was absent in 60% of the tested mice after the injection (Figure 16a). The onset time for the effect of pentobarbital (40 mg/kg) on their pinprick responses was found to be about 3 minutes. The performance of all mice on the rotarod was impaired for about 45 minutes (Figure 16b). Again, the righting reflexes of all of the mice in this group were lost during this period. As soon as the mice regained their righting reflexes, they regained their abilities of walking on the rotarod very rapidly. Notably in these experiments, tail flick responses to pin-prick were observed in mice whose performance on the rotarod was completely suppressed (Figure 16) 7 9 Position D (n = 5) c •B c o Q . . O tn i -O Q. «- c CD ' Q . 100 80-60 -4 0 20-- * - Pentobarb (30mg/kg) -o - Pentobarb (40mg/kg) —i 1 1 1 1 1 1 10 20 30 4 0 50 6 0 70 Time (min) Performance of Mice on the Rotarod (n = 5) (a) T3 O ns + J o OH _ c o ° s o ro 3 Q 10i> 9 8 7 6 5 4 3 2 1 0 I J t •YYYY A Pentobarb (30mg/kg) o Pentobarb (40mg/kg) 5* 0 10 2 0 30 4 0 50 Time (min.) 60 7o (b) Figure 16: The effects of intraperitoneal injection of pentobarbital (30 mg/kg, 40 mg/kg) on (a) the pinprick response and (b) the performance on a rotarod of the mice. The drug was injected at t=0 minute. The sample size = 5 mice per group. For the graph (b), each point represents the averaged duration (±SEM) of each group of mice on the rotarod at the corresponding time. 80 3.2.1.4.Rocuronium (0.7 mg/kg) Intraperitoneal injection of a high dose (close to LD 5 0 ) of rocuronium (0.7 mg/kg, 0.5 ml) into a group of normal mice (n=5) had no effect on their tail-flick responses to pinprick, even though the ability of the mice to walk on the rotarod was impaired. Among the 5 mice tested, 3 died within 6 minutes after the injection. These mice were unable to walk on the rotarod at about 2 minutes after the drug was injected. However, the tail-flick response to pinprick was not affected in any of these 3 mice until the mice stopped breathing, which was about 1 minute before the mice died. None of these mice were able to walk on the rotarod at 2 minutes after the drug was injected. For the 2 mice that survived, the pinprick response of one of them was briefly blocked for about 5 minutes when it was twitching in the Plexiglas mouse-holder. The pinprick response of the other surviving mouse was not blocked at any time after the drug was injected. The performance on the rotarod of both surviving mice was significantly (P<0.05) impaired at about 3 minutes after injection for about 12 minutes and 18 minutes after that point. 81 3.2.2. The Effects of Systemic Administration (with the Tail Occluded by a Tourniquet) of Analgesic Drugs on Pinprick Pain in Normal Mice 3.2.2.1.Lidocaine (75 mg/kg) In this experiment, lidocaine (75 mg/kg, 0.5 ml) was injected intraperitoneally into a group of mice (n=5) immediately after the application of a tourniquet around the base of their tails. Since the blood flow to the tail was occluded by the tourniquet, the hypothesis was that if the mice did not respond to pinprick after the injections, the analgesia induced was due to the action of the drug on sites that were not located within the tail. The tourniquet was applied for only 20 minutes, which was determined from the experimental results described in section 3.1.2. It was found that the injection of lidocaine induced a statistically significant (P<0.05) effect of their tail-flick responses to pinprick at both positions D and P during and after the tourniquet period (Figure 17). On the other hand, it was found that the injected saline (0.5 ml, ip) into the control group of mice (n=5) had no effect on their pinprick responses and their performance on a rotarod. The onset of the effect if lidocaine (75 mg/kg, ip) of the pinprick response at position D was approximately 3 minutes after the drug was injected, after which no pinprick responses of the mice in this group were observed during the tourniquet period. The absence of their tail-flick responses to pinprick continued for another 20 minutes after the tourniquet was released; one of these mice did not respond to the pinprick test for another 140 minutes. Position P was located proximal to the tourniquet-site. The duration of the 82 absence of tail-flick response to pinprick at this position was found to be longer than that at position D. The onset of the effect of lidocaine (75 mg/kg, ip) was approximately 2 minutes after the drug was injected. The pinprick responses were absent during the rest of the tourniquet period. The mice were not responding to the pinprick test for another 40 minutes, approximately, after the tourniquet was released, except for one mouse that did not respond to the pinprick test for another 140 minutes. Compared to the control group into which saline was injected, the rotarod performance of the mice that received lidocaine (75 mg/kg, ip) was also significantly (P<0.05) affected (Figure 17b). 10 minutes after the drug was injected, the averaged duration on the rotarod of all the mice in this group dropped to 0 second. Then, the mice gradually regained their ability to walk on the rotarod. At 70 minutes after the drug was injected, all mice were capable of walking, on the. rotarod for 10 seconds. However, the ability of the mice to walk on the bench and their spontaneous tail movements were not affected throughout the experiment. The results indicated that the absence of the tail-flick responses to pinprick could be attributed to an analgesic action of lidocaine rather than any action on the motor system. Also, since the blood flow to the tail was occluded, the results indicated that the analgesia induced by lidocaine could not be due to an action of lidocaine at sites located within the tail. 83 Positions D and P - Position D • Position P 0-0-6 I I I 1 1 I 1 1 0 20 4 0 6 0 80 100 120 140 160 180 Time (min) The performance of the mice on a rotarod T3 O 1_ CO o c o ° % o co 1_ 3 Q 10 8 6H • i — i — i — i — i — i — i — i — i 0 2 0 4 0 6 0 80 100 120 140 160 180 Time (min) (a) (b) Figure 17: The effects of intraperitoneal injection of lidocaine (75 mg/kg, 0.5 ml) immediately after a tourniquet was applied on normal mice (n=5). The drug was injected at t=0 minute. The tourniquet was released at t=20 minutes, (a) The responses of the mice to pinprick at positions D and P. (b) The performance of the mice on a rotarod. Each point on graph (b) represents the averaged duration of the mice on a rotarod (±SEM) at the corresponding time. 84 3.2.3. The Effects of Peripheral Administration of Analgesic Drugs on Pinprick Pain in Normal Mice In this set of experiments, each mouse was first anaesthetized with 2% isoflurane in a 5L anaesthetic chamber. One of the test drugs of volume 0.05 ml was injected intravenous into the mouse tail (distal to the tourniquet site) immediately after a tourniquet was applied around the tail at a site where the tail diameter was 3.5 mm. The mouse was then removed from the anaesthetic chamber. After that, pinprick test was immediately performed. The duration of the tourniquet was 15 minutes. The drugs tested were morphine (1.5%), lidocaine (1%), bupivacaine (0.3%), QX-314 (2%), and A S A (2.4%) and saline control (0.05 ml). Injection of saline (control) had no effect on the pinprick response of any mouse in the control group (n=5). 3.2.3J.Morphine (1.5%) During the 15-minute tourniquet period, the injected morphine (1.5%) induced an inconsistent effect on the tail-flick response to pinprick at position D of 60% of the mice in this group (n=5). That is, the mice responded inconsistently to the pinprick test at this site. However, as the tourniquet was released after it had been applied for 15 minutes, the morphine that was previously trapped in the tail was released into the circulation. This was equivalent to releasing an intravenous dose of 25 mg/kg morphine into the circulation. Within 3 minutes after the tourniquet was released, all the mice in this group stopped responding to the pinprick test. The absence of their pinprick responses persisted for at least another 200 minutes in all the mice. The pinprick test was paused 85 at this time, and the mice were checked again on the next day. On the 2 n d day of the experiment (24 hours after the drug was injected), all the mice in this group were responding to pinprick test by withdrawing their tails again. 3.2.3.2.Lidocaine (1%), Bupivacaine (0.3%), and QX-314 (2%) Intravenous injections of 1% lidocaine, 0.3% bupivacaine, and 2% QX-314 into the tails of normal mice distal to the tourniquet-site was found to be effective in blocking their tail-flick responses to pinprick at position D. The volume of injection was 0.05 ml, and the drug was injected at t=0 minute. For the group of mice that received 1% lidocaine (n=5), during the 15-minute duration of tourniquet application, none of the mice responded to pinprick at position D. The absence of their pinprick responses continued for about 15 minutes after the tourniquet was released. At t=40 minutes, 100% of the mice were responding to pinprick at position D positively again. For the group of mice that received 0.3% bupivacaine (n=5), all of the tested mice were not responding to the pinprick at position D on their tails during the 15-minute tourniquet period. The absence of their pinprick responses continued for another 20 minutes after the tourniquet was released. At t=60 minutes, all the mice in this group were responding to the pinprick at position D positively again. Injection of 2% QX-314 induced a prolonged effect on the pinprick response of all that mice that received this drug (n=5). The onset time of the effect of 2% QX-314 was approximately 3 minutes. All the mice in this group were not responding to pinprick 86 test at position D on their tails throughout the tourniquet period. The absence of their pinprick responses continued for the next 5 hours after the drug was injected. On the 2 n d day of the experiment (24 hours after the drug was injected), all the mice in this group were responding to pinprick test by withdrawing their tails again. However, for these 3 groups of mice, their responses to pinprick at position P were not affected at any time during the experiment. This suggested that QX-314 acted like lidocaine and bupivacaine when administered peripherally. They were capable of blocking the perception of pinprick pain by locally blocking the peripheral pain fibres without affecting the sites that they could not reach (e.g. position P). 3.2.3.3.ASA (2.4%) Intravenous injection (distal to the tourniquet-site) of 2.4% A S A (0.05ml) had no effect on the tail-flick responses to pinprick (at positions D and P) of the mice (n=5) throughout the first 300 minutes after the drug was injected. Also, all the mice in this group were responding to pinprick test by flicking their tails during the 6-day follow-up period. 87 3.3. Phase III: Effects of Standard Analgesic Agents on Tourniquet-Induced Post-ischaemic Allodynia in Mice As seen in the previous section, most of the analgesic/anaesthetic drugs were able to block pinprick pain. This raised the question of whether these drugs were able to block the appearance of post-ischaemic allodynia, which is the focus of the current study. In this set of experiments, the effects of various analgesic/anaesthetic agents on tourniquet-induced post-ischaemic allodynia were tested. 3.3.1 The Effects of Systemic Administration of Analgesic Drugs on Tourniquet-Induced Post-ischaemic Allodynia in Mice In this set of experiments, post-ischaemic allodynia in the mouse tails was induced by a 60-minute tourniquet, which guarantees prolonged allodynia (see above). The presence of allodynia in a mouse tail was assessed by touching the tail with a piece of V F H . After allodynia had been observed in the mouse tails for 30 minutes, one of the test drugs was injected subcutaneously at the back of a mouse neck. The volume of injection was 0.5 ml. The drugs tested were morphine (5mg/kg, 10 mg/kg, 20 mg/kg), lidocaine (75 mg/kg), bupivacaine (50mg/kg), QX-222 (140 mg/kg), pentobarbital (30 mg/kg, 40 mg/kg), rocuronium (0.7 mg/kg) 3.3.1.1.Morphine (5 mg/kg, 10 mg/kg, 20 mg/kg) Subcutaneous injection (at the back of the neck) (sc) of morphine (5 mg/kg, 10 mg/kg, 20 mg/kg) into allodynic mice induced a dose-dependent effect on their 88 withdrawal responses to touch at their tails (Figure 18). For the group of mice that received 5 mg/kg morphine (sc) (n=5), the tail-flick responses to the touch of a piece of V F H was absent in 40% of the tested mice after the injections. However, all the mice that received 10 mg/kg (sc) (n=5) and 20 mg/kg (sc) (n=5) of morphine (sc) stopped responding to touch for durations of 90 minutes and 110 minutes, respectively, after the injections. For these groups of mice, ability to walk on the experiment bench and spontaneous tail movements were not affected during the experiment, indicating that the mice were not paralysed in the experiment. 89 The effect of various doses of morphine on allodynic mice at position D 100 CN X 80 > O +J O) c 6 0 T3 C o a w o CJ) u 4 0 2 0 —A <k-V -V--/ / / / / / / / - V , I I I I y d v v v v y 30 60 90 120 150 Time (min) — M o r p h i n e 5 mg/kg Morphine 10 mg/kg Morphine 20 mg/kg (a) 180 Figure 18: The effects of subcutaneous injection (at the back of a mouse neck) of various doses of morphine on the tourniquet-induced post-ischaemic allodynia in mice. Post-ischaemic allodynia was induced by a 60-minute tourniquet in all 3 groups of mice (n=5 mice per group). Morphine was injected at t = 0 minute, at which time allodynia had been observed in the tails for 30 minutes. The effects of morphine on allodynia at positions D and P are shown in graphs (a) and (b) respectively. 90 The effect of various doses of morphine on allodynic mice at Position P -| v » w y « v v v v y v y v 1 1 1 0 30 6 0 90 120 150 180 Time (min) —*— Morphine 5 mg/kg - - V - - Morphine 10 mg/kg Morphine 20 mg/kg (b) 91 3.3.1.2.Lidocaine (75 mg/kg), QX-222 (140 mg/kg), and Bupivacaine (50 mg/kg) The tail-flick response of the allodynic mice to the touch of a V F H was significantly (P<0.05) affected by systemic injection of lidocaine (75 mg/kg) (Figure 19a) and bupivacaine (50 mg/kg) (Figure 19b), but not QX-222 (140 mg/kg). The drug was injected subcutaneously into the back of the neck at t=0 minute. The onset of the effect of lidocaine (75 mg/kg, sc) on the tail-flick responses to touch of a group of mice (n=5) was found to be approximately 15 minutes after the injection. An absence of tail-flick responses to touch was observed in all mice at t=22 minutes after injection. At t=45 minutes, the percent of mice that were responding touch at position D increased. At t = 90 minutes, all the mice responded positively to touch (at position D) again by flicking their tails. For the group of mice that received bupivacaine (50 mg/kg) (n=5), the onset of action of the injected bupivacaine was approximately 3 minutes. The absence of the tail-flick responses to touch at position D was observed in all the mice in this group at t=5 minutes. At t=20 minutes, the percentage of mice that were responding to touch at this position gradually increased. At t = 130 minutes, all the mice responded positively to the touch of the V F H (at position D) again by flicking their tails. The injection of QX-222 (140 mg/kg) into another group of allodynic mice (n=20) had no effect on their tail-flick responses to the touch of a piece of a V F H at position D on their tails. In these 3 groups of mice, their spontaneous tail movements and their abilities of walking on the experiment bench were not affected. Also, these mice were able to breathe spontaneously and an artificial respirator was not required. Therefore, it was 92 unlikely that the injected lidocaine, bupivacaine (and QX-222) blocked the motor function of these mice. 93 100 100-1 CO c O CM C a t CO *- L L CD > O Position D (Lidocaine 75 mg/kg) 30 6 0 90 Time (min) Position D (Bupivacaine 50 mg/kg) 120 60 90 120 150 180 Time (min) (a) (b) Figure 19: The effects of subcutaneous injection (at the back of a mouse neck) of (a) 75 mg/kg lidocaine and (b) bupivacaine (50 mg/kg) on tourniquet-induced post-ischaemic allodynia in mice (n=5). Allodynia was induced by a 60-minute tourniquet. The drug was injected at t=0 min, at which time allodynia at position D had been observed for 30 minutes. 9 4 3.3.1.3.Pentobarbital (30 mg/kg, 40 mg/kg) Intraperitoneal injection of 40 mg/kg, but not 30 mg/kg, of pentobarbital was found to have a significant (P<0.05) effect on the tail-flick responses of the allodynic mice to the touch of a V F H . Compared to the saline control group, the performance of both groups of mice (received pentobarbital ip) on a rotarod was significantly (P<0.05) affected. Intraperitoneal injection of 30 mg/kg pentobarbital into a group of mice (n=5) had no effect on their tail-flick responses to touch at position D. For the group of mice that received 40 mg/kg pentobarbital intraperitoneally (n=5), tail-flick responses to touch at position D were absent for about 27 minutes after the injections, with an onset time of approximately 5 minutes (Figure 20a). The drug was injected at t=0 minute. The tail-flick responses of all these mice were absent from t=10 minutes to t=20 minutes. Then, the tail-flick responses were gradually found again in this group of mice. At t=50 minutes, all the mice (received 40 mg/kg pentobarbital) responded positively to the touch of a von Frey filament again. The performance of the allodynic mice on a rotarod was affected by intraperitoneal injection of pentobarbital in a dose-dependent manner (Figure 20b). For the group of mice that received 30 mg/kg pentobarbital (ip), their averaged duration on a rotarod dropped to 0 second at t=10 minutes. At this time, all the mice in this group were sedated and their righting reflexes were lost. This group of mice began to regain their righting reflexes and their ability to walk on the rotarod at t=30 minutes. At t=60 minutes, all the mice were able to walk on the rotarod for 10 seconds again. For the group of mice that received 40 mg/kg pentobarbital (ip), their averaged 95 duration on the rotarod dropped to 0 second at about t=10 minutes. At this time, all the mice in this group were sedated and their righting reflexes were lost. This group of mice began to regain their ability to walk on the rotarod at t=40 minutes. At t=90 minutes, all the mice were able to walk on the rotarod for 10 second again. 3.3.1.4.Rocuronium (0.7 mg/kg) Intraperitoneal injection of 0.7 mg/kg had no effect on the tail-flick responses of the allodynic mice to touch at both positions D and P, even though the performance of these mice on a rotarod was slightly impaired for about 20 minutes after the injection (Figure 21). 96 Position D O) p c O CM LL O > O 100 80 HI 6 0 4 0 2 0 4 0 - * * A * A 40 mg/kg i 1 1 r——i 0 2 0 4 0 6 0 80 100 c o c .2 "o 0 (0 2 k . i 10i> 8 6 4 -2 -0 Time (min) Performance of mice on a rotarod o © • (a) o 30 mg/kg * 40 mg/kg -o—a-0 2 5 50 75 100 Time (min) (b) Figure 20: The effects of intraperitoneal injection of pentobarbital (30 mg/kg, 40 mg/kg) on (a) tourniquet-induced post-ischaemic allodynia (at position D) in mice and (b) their performance on a rotarod. The drug was injected at t=0 minute. Sample size = 5 mice per group. For graph (b), each point represents the averaged duration (±SEM) of each group of mice on a rotarod at the corresponding time. 97 Performance on Rotarod 10 O 1_ ro •*-> O i _ c 'u O CD o '*z ro 3 Q —I— 15 Time (min) Rocuronium 0.7mg/kg 30 Figure 21: The effect of intraperitoneal injection of rocuronium (0.7 mg/kg) on the performance on rotarod of allodynic mice (n=5). 3 out of 5 mice died after the injection of the drug. Each point represented the averaged duration (±SEM) on the rotarod of the 2 surviving mice. The drug was injected at t=0 minutes, at which time allodynia had been observed in the tails for 30 minutes. 98 3.3.2. The Effects of Systemic Administration (with tail Occluded by a Tourniquet) of Analgesic Drugs on Tourniquet-Induced Post-ischaemic Allodynia in Mice In previous sections, the effects of various analgesic agents on tourniquet-induced post-ischaemic allodynia were described. These drugs could have blocked the appearance of allodynia in the mice through their actions on the C N S and/or the P N S . The results from previous experiments did not show if the systemically injected analgesic drugs were acting on the C N S or the peripheral nociceptive nerve fibres. In this experiment, we occluded the blood flow to the tails of the allodynic mice before the analgesic drugs were injected systemically. Consequently, whether the injected analgesic drugs possessed sites of actions that were not located in the tail (distal to the tourniquet-site) could be investigated. In this set of experiments, post-ischaemic allodynia in the mouse tails was induced by the application of a 30-minute tourniquet around the base of the tails. The presence of allodynia in the tails was assessed by touching the tail with a V F H . After allodynia had been observed in the mouse tails for 30 minutes, the allodynic mice were anaesthetized with 2% isoflurane by placing them into a 5L anaesthetic chamber. For each allodynic mouse, a second tourniquet was immediately applied around the tail at a site where the tail diameter was 3.5 mm. This was followed by intraperitoneal injection of one of the test drugs. The volume of injection was 0.5 ml. After the injection, the mouse was immediately removed from the anaesthetic chamber. The duration of the second tourniquet was 20 minutes. The presence of allodynia in the tail was, again, assessed by touching the tail with a V F H . The following drugs were tested in this set of experiments: morphine (10 mg/kg, 20 mg/kg), lidocaine (75 mg/kg), and saline control. 9 9 The injection of saline had no effect on their tail-flick responses to touch at either position D or P in the control group of mice (n=5). 3.3.2.1.Morphine (10 mg/kg, 20 mg/kg) Intraperitoneal injection of morphine (10 mg/kg, 20 mg/kg) into allodynic mice (with the blood flow to the tail occluded by a tourniquet) induced a dose-dependent effect on the tourniquet-induced post-ischaemic allodynia in the tails (Figure 22a). Morphine (10 mg/kg, 20 mg/kg) was injected immediately following the application of the tourniquets around the tails of two groups of anaesthetized allodynic mice (n=5) at t=0 minute. During the 20-minute tourniquet period, the tail-flick responses to touch at position D were absent in all the mice in the group that received 10 mg/kg morphine intraperitoneally. The onset time was found to be approximately 3 minutes. The absence of their tail-flick responses to touch continued for approximately 100 minutes after the tourniquets were released. The performance of all the mice in this group on the rotarod was found to be slightly impaired for approximately 60 minutes after injection (Figure 22b). Results of the V F H test at position P for both group of allodynic mice were found to be similar to those observed at position D. For the group of allodynic mice that received 20 mg/kg morphine, the tail-flick responses to touch at position D were absent in all the mice in this group with an onset time of 3 minutes; none of the mice responded to the touch of the V F H throughout the rest of the 20-minute tourniquet-period. Their tail-flick responses to touch continued to be absent for approximately 200 minutes after the tourniquets were released. The 100 performance of all the mice in this group on the rotarod was found to be moderately impaired for approximately 220 minutes after injection (Figure 22c), but their abilities of walking on the bench and their spontaneous tail movements were not affected during this period. The average duration on a rotarod dropped to 2 seconds at t=2 minutes. However, at about t=80 minutes, the mice began to regain their abilities to walk on the rotarod. At t=240 minutes, all the mice were able to walk on the rotarod for 10 seconds again. These results indicated that morphine blocked the appearance of allodynia through action(s) on site(s) that were not located locally in the tails. These sites could possibly be located in the C N S and they were discussed in section 1.4.3.1. 101 Position D 0 100 1 80-c O CN g"* 6 0 4 8> 4 0 -E O 2 0 -0 Morphine 10 mg/kg Morphine 20 mg/kg o A A A A A A A A A j 60 120 180 240 Time (min) Position P (a) 100 80 c S c O CM 604 L i . 8> 40-20-o l A--A Morphine 10 mg/kg Morphine 20 mg/kg A A A A A A A A A A* 60 120 Time (min) 180 240 (b) Figure 22: (a, b) The effects of ip injection (with the blood flow to the tails occluded) of morphine (10 mg/kg, 20 mg/kg) on tourniquet-induced post-ischaemic allodynia in mice. Allodynia was induced by a 30-minute tourniquet in both groups (n = 5). The drug was injected at t=0 minute, at which time allodynia had been observed at position D for 30 minutes, (c) The effects of the morphine on the rotarod performance of the allodynic mice. Each point on graph (c) represents the averaged duration (±SEM) of each group of mice on a rotarod at the corresponding time. 102 Performance of mice on rotarod 10-f 84 4H 2H Morphine 10 mg/kg Morphine 20 mg/kg 60 120 Time (min) 180 240 (C) 103 3.3.2.2.Lidocaine (75 mg/kg) With the circulation to the tails occluded by a tourniquet, intra-peritoneal injection of 75 mg/kg lidocaine into a group of allodynic mice (n=5) was found to be effective in affecting their tail-flick response to touch, for approximately 70 minutes after injection (Figure 23). Lidocaine (75 mg/kg) was injected immediately following the application of the tourniquets around the tails of the anaesthetized allodynic mice. During the 20-minute tourniquet-period, the tail-flick responses to touch at position D were absent in all these mice with an onset time of approximately 3 minutes. The absence of their tail-flick responses to touch continued for approximately 40 minutes after the tourniquets were released. The responses to touch at position P of this group of allodynic mice were found to be very similar to that at position D. Although the performance of all mice in this group on a rotarod was significantly (P<0.05) impaired, their spontaneous tail movements and their ability to walk on the rotarod was not affected during this period. These results indicated that lidocaine induced analgesia though an action or actions on site(s) that were not located in the allodynic mouse tail (distal to the tourniquet-site). These sites could be located in the central nervous system, but they have not yet been identified. This will be further discussed in the section 4.4.2. 104 Positions D and P O 100-r D) c c O C N g-» 60 *" L L 0) > O Position D Position P Time (min) Performance on Rotarod (a) 1(W T3 O ro 4-1 o c 7> o CJ) c «L o co 3 Q 30 60 Time (min) Lidocaine (75mg/kg) —i 90 (b) Figure 23: The effects of lidocaine (75 mg/kg, ip) (with blood flow to the tails occluded) on tourniquet-induced post-ischaemic allodynia in mice (n=5). The effects of lidocaine on allodynia at positions D and P are shown in graph (a), (b) The effects of lidocaine on the rotarod performance of the same group of allodynic mice. The drug was injected at t=0 minutes, at which time allodynia had been observed in the tails for 30 minutes. Each point on graph (b) represents the averaged duration (±SEM) of each group of mice on a rotarod at the corresponding time. 105 3.3.3. The Effects of Peripheral Administration of Standard Analgesic Agents on Tourniquet-Induced Post-ischaemic Allodynia In this set of experiments, post-ischaemic allodynia in the mouse tails was induced by the application of a 30-minute tourniquet around the base of the tails. The presence of allodynia in the tails was assessed by touching the tail with a V F H . After allodynia had been observed in the mouse tails for 30 minutes, the allodynic mice were anaesthetized with 2% isoflurane by placing them into a 5L anaesthetic chamber. For each allodynic mouse, a 300 mmHg pressure cuff was applied around the base of its tail. This was followed by intravenous injection of one of the test drugs at a site that was close to the tip of the tail. The volume of injection was 0.05 ml. A second tourniquet (plastic tubing) was immediately applied around the tail at a site where the tail diameter was 3.5 mm. After that, the mouse was immediately removed from the anaesthetic chamber. The duration of the second tourniquet was 15 minutes. The presence of allodynia in the tail was, again, assessed by touching the tail with a V F H . The following drugs were tested in this set of experiments: morphine (1.5%), lidocaine (1%), bupivacaine (0.3%), QX-314 (2%), and saline control. The injection of saline into the control group of mice (n=5) had no effect on their tail-flick responses to touch at either position D or P. 3.3.3.1.Morphine (1.5%) During the 15-minute tourniquet period, only one of the mice (n=5) that received morphine (1.5%, local iv) stopped responding to the touch of a piece of V F H at position 106 D, and inconsistently (Figure 24a). The responses of the mice to touch at position P were not affected by the injected morphine in this period. However, as the tourniquet was released after it had been applied for 15 minutes, the morphine that was previously trapped in the tail was released into the circulation. This was equivalent to releasing an intravenous dose of 25 mg/kg morphine into the circulation. Within 3 minutes after the tourniquet was released, none of the mice in this group responded to touch at either position D or P on their tails. The absence of their tail-flick responses to touch persisted for at least another 200 minutes in all the mice. The testing for allodynia was paused at 200 minutes after the drug was injected, and it was continued to be performed on the next day of the experiment. On the 2 n d day of the experiment, all the mice in this group were found to be responding to touch at positions D and P by flicking their tails again. 3.3.3.2.Lidocaine (1%), Bupivacaine (0.3%), and QX-314 (2%) For the group of allodynic mice that received lidocaine (1%, local iv) (n=5), none responded to touch at positions D during and after the tourniquet period (Figure 24b). The onset of action of lidocaine (1%) on the appearance of allodynia at position D was approximately 2 minutes after the drug was injected. The tail-flick responses of the all the allodynic mice to touch were absent for the rest of the tourniquet period (15 minutes). The absence of their responses to touch continued for approximately 70 minutes after the tourniquets were released. Of the group of allodynic mice that received bupivacaine (0.3%, local iv) (n=5), none responded to touch at position D during and after the tourniquet period (Figure 107 24c). The onset of action of bupivacaine (0.3%) was approximately 2 minutes after the drug was injected. The tail-flick responses of the all the allodynic mice (n=5) to touch were absent for the rest of the tourniquet period (15 minutes). The absence of their responses to touch for approximately 90 minutes after the tourniquet was released. The injection of QX-314 (2%, local iv) induced a prolonged effect on the tail-flick responses of the allodynic mice to touch at position D. The onset of the action of QX-314 was approximately 2 minutes after the drug was injected. The tail-flick responses of the all the allodynic mice (n=5) to touch were absent for the rest of the tourniquet period (15 minutes). The absence of their responses to touch continued for at least another 285 minutes after the tourniquets were released. All the mice in this group were found to be responding to the V F H test positively again on the 2 n d day of the experiment. For the above 3 groups of mice, all of them responded to the touch at position P by flicking their tails, at any time during the experiment. 108 Morphine (1.5%) c S c O CN V) * *- u_ d) > o 100<*OOOOOOOO 80 60 40 — Position D -o-- Position P 10 • — • • — • — ^ -20 30 40 50 60 Time (min) (a) o 4-> Lidocaine (1%) 100 ^OC<XXXXXXXXXXXX><><> O -0 o 4» » » » » » » —— Position D o Position P 30 60 Time (min) (b) Figure 24 The effects of peripheral local injections of (a) morphine (1.5%), (b) lidocaine (1%) and (c) bupivacaine (0.03%) on tourniquet-induced post-ischaemic allodynia in mice. The drugs were injected at t=0 minute immediately following the application around the base of the allodynic mouse tails. The tourniquet was released at t=15 minutes. 1 0 9 Bupivacaine (0.03%) 100-»OCC<XXXXXXXXXXX>0-0-0--0-0 o-o <>--• » » » Time (min) —— Position D o Position P (c) 110 3.4. Phase IV: Effects of Standard Analgesic Agents on the Development of Tourniquet-Induced Post-ischaemic Allodynia in Mice 3.4.1 The Effects of Systemic Administration of Analgesic Agents on the Development of Tourniquet-Induced Post-ischaemic Allodynia in Mice As seen in previous sections, some of the analgesic drugs were effective in blocking the appearance of allodynia in mice, but none of these drugs permanently abolished the induced allodynia. In this set of experiment, we investigated if the development of allodynia might be blocked by blocking the pain experienced by the mice during the tourniquet period. One of the test drugs was injected ip immediately after the application of a tourniquet around the base of a mouse tail. The duration of tourniquet used in the experiments was 30 minutes. The drugs tested were morphine (10 mg/kg, 40 mg/kg), lidocaine (75 mg/kg), A S A (100 mg/kg, 300 mg/kg, 400 mg/kg) and saline control. 3.4.1.1.Morphine (10 mg/kg, 40 mg/kg) and Lidocaine (75 mg/kg) Intra-peritoneal injection of morphine (10 mg/kg) or lidocaine (75 mg/kg) immediately prior to the application of a tourniquet around the base of a mouse tail was found to be ineffective in preventing the development of allodynia in the tail. For the group of mice that received 10 mg/kg morphine, the appearance of allodynia was delayed but it was detected in 100% of this group of mice at both positions at about 70 minutes after the tourniquets were released. For the group of mice that received 75 mg/kg lidocaine, the appearance of allodynia was also delayed and it was detected in 111 100% of this group of mice at both positions at about 40 minutes after the tourniquets were released. These results raised the question whether the injected drugs masked the appearance of the induced allodynia or delayed its development? In order to answer this question, another experiment was performed, in which morphine (40 mg/kg) and lidocaine (75 mg/kg) were tested. The drugs were injected intraperitoneally immediately after the application of a tourniquet around the mouse tails. The duration of the tourniquet used was 30 minutes. In this experiment naloxone (10 mg/kg) was injected at 10 minutes after the tourniquet was released. Of the group of mice that received morphine (40 mg/kg, ip) (n=5), none responded to the touch of a piece of V F H during and for the first 10 minutes after the tourniquet-period. However, within 5 minutes after naloxone (10 mg/kg) was injected intraperitoneally, all the mice were found to respond to touch by flicking their tails. This suggests that allodynia had already developed in the mouse tails during the tourniquet period. The injected morphine only delayed the appearance of the induced allodynia. Otherwise, the onset of allodynia would be similar to that observed in Figure 5. On the other hand, of the group of mice that received lidocaine (75 mg/kg, ip), none responded to touch with a piece of V F H during and after the first 10 minutes after the tourniquet period; the injection of naloxone (10 mg/kg, ip) had no effect on the absence of the tail-flick response to touch in this group of mice. The mice were not responding to touch at those tails for about another 15 minutes. The duration of the absence of the tail-flick responses of this group of mice was not shortened by the 112 injection of naloxone (10 mg/kg), indicating that the actions of lidocaine on the perception of the induced allodynia was not naloxone reversible. 113 Position D i i l l l l l l l l l l l l l l l i l l lHHBBfr ^ 1 1 1 1 1 1 1 0 30 60 90 120 150 180 210 240 270 300 Time (min) —•— Saline Control - a - Morphine 10 mg/kg - A - - - Lidocaine 75 mg/kg (a) Figure 25 The effects of intraperitoneal injections of morphine (10 mg/kg), lidocaine (75mg/kg) and saline control (0.5ml) on the development of allodynia in mice (n=5). The duration of the tourniquet used in this experiment was 30 minutes. The drug was injected immediately prior to the application of the tourniquets around the mouse tails. Graphs (a) and (b) show the responses of the mice to the touch of a piece of VFH at positions D and P respectively. On the graphs, t=0 minute indicates the time at which the drugs were injected and t=30 minutes indicates the time at which the tourniquets were released. 114 Position P 100n CN tt Time (min) —•— Saline Control -a- Morphine 10 mg/kg Lidocaine 75 mg/kg (b) Positions D and P Time (min) —— lidocaine (75 mg/kg) --o-- morphine (40 mg/kg) Figure 26 The effects of morphine (40 mg/kg) and lidocaine (75 mg/kg) on the development on tourniquet-induced post-ischaemic allodynia in mice. Morphine or lidocaine was injected ip at t=0 minute, immediately following the application of a tourniquet around the mouse tail. The tourniquet was released at t=30 minutes. Naloxone (10 mg/kg) was injected at t=40, indicated with an arrow in the graph. The responses of the two groups of mice (n=5) to touch at positions D and P were the same, so that the results are shown on the same graph. 116 3.4.L2.ASA (400 mg/kg, 300 mg/kg, and 100 mg/kg) Intra-peritoneal injection of A S A (400 mg/kg, 300 mg/kg, and 100 mg/kg) 30 minutes prior to the application of the tourniquet was found to be ineffective in preventing the development of post-ischaemic allodynia in mice induced by a 20-minute tourniquet (n=5 for each dose). All (100%) of the mice that received A S A (400 mg/kg, 100 mg/kg), or saline (control) developed allodynia at position D at 110 minutes, 4 minutes, and 4 minutes after the tourniquet was released respectively. 80% of the mice that received 300 mg/kg A S A developed allodynia at position D after the tourniquets were released. During the 6-day follow-up period, 2 mice in the group that received 400 mg/kg A S A (ip) recovered completely from the induced allodynia at positions D and P on the 3 r d day of the experiment, while the rest of the group remained allodynic at both positions on the 6 t h day of the experiment. For the group of mice that received 300 mg/kg A S A (ip), 1 out of the 4 allodynic mice recovered from the induced allodynia at positions D and P on the 3 r d day of the experiment. The other 3 mice were still allodynic at both positions D and P on the 6 t h day of the experiment. Of the group of mice that received 100 mg/kg A S A (ip), 40% and 60% of the group recovered from the induced allodynia at either position D or P, respectively, at the end of the 6-day follow-up period. The performance on a rotarod of the mice, which received 400 mg/kg or 300 mg/kg A S A intra-peritoneally, was significantly affected by the drug. For the group of mice that received 400 mg/kg A S A , the averaged duration on the rotarod was reduced to 5 sec at the time when the tourniquet was released. They gradually regained their abilities to walk on the rotarod afterwards. At 180 minutes after the tourniquet was 117 released, all the mice in this group were able to walk on the rotarod for 10 seconds again. The effects of 300 mg/kg A S A on the rotarod performance of the mice were found similar to when 400 mg/kg A S A was injected. However, the performance of the mice that received saline (control) or 100 mg/kg A S A was not affected at all. In this experiment, it was found that the injections of A S A neither blocked nor delayed the appearance of allodynia in the mice, suggesting that the prostaglandins that might have been produced during the ischaemia in the mouse tails might not be an important factor contributing to the development of allodynia in the mice. 118 Position D (First 180 Minutes) CM tt I > o 100", cn c c o Q. (0 0) 1 _ o u 80 5 60 40 = 20- i O 04 (ccccccco- o - o- -o - o - o- - o - -o -o- ® ® - ® — ® t——*.—4—*—A — A S A 400 mg/kg -* - A S A 300 mg/kg - o - A S A 100 mg/kg —i— 30 60 90 120 150 180 Time (min) (a) Figure 27 The effects of various doses of ASA (100 mg/kg, 300 mg/kg, and 400 mg/kg) on the development of tourniquet-induced post-ischaemic allodynia in mice. The drugs were injected intraperitoneally into the mice 20 minutes prior to the application of a 30 minutes tourniquet around the base of their tails. Sample size for each group of mice = 5. Graphs (a) and (b) show the responses of the mice to touch at positions D and P, respectively, in the first day of the experiment. Graphs (c) and (d) show their responses to touch at positions D and P in the 6-day follow-up period. In the graphs, t=0 minute indicates the time at which the tourniquets were released. 119 Position P (First 180 Minutes) 120 Position D (6-Days Follow-Up Period) — A S A 400 mg/kg A S A 300 mg/kg - o - A S A 100 mg/kg 120 Time (hours) Position P (6-Days Follow-Up Period) Time (hours) — A S A 400 mg/kg • A S A 300 mg/kg - o - A S A 100 mg/kg 120 (d) 3.4.2. The Effects of Inhalation General Anaesthetics - Isoflurane - on the Development of Tourniquet-Induced Post-lschaemic Allodynia in Mice In this experiment, we investigated if the conscious perception of pain of the mice was essential in the development of tourniquet-induced post-ischaemic allodynia in their tails. The animals were generally anaesthetized with 2% isoflurane for 30 minutes before the tourniquet was applied, during the 30-minute tourniquet period, and for 30 minutes after the tourniquet was released. During the anaesthesia period, the withdrawal reflexes to pinprick at the hind legs as well as at the tails of all mice were lost. Also, all these mice had lost their righting reflexes during this period. Generally anaesthetizing the animals was found to be ineffective in blocking the development of post-ischaemic allodynia in mice (n=5), but the onset of the allodynia (or its appearance) was delayed. Allodynia was detected in 100% of the mice in this group about 30 minutes after they were removed from the anaesthetic chamber. In Figure 28a, the mice were removed from the anaesthetic chamber at t=0 minutes. The mice began to regain their righting reflexes at about t=20 minutes, and all mice were responding to the touch with a piece of V F H at t=36 minutes. When the mice were removed from the anaesthetic chamber, they were still anaesthetized; their righting and withdrawal reflexes were lost. As soon as they began to regain their righting reflexes at about t=20 minutes, they began to walk on the experiment bench and respond to the touch stimulus. The duration of the delay of the appearance of allodynia in this group of mice was about the same as the time required for the normal mice to regain their withdrawal reflexes toward a painful stimulus, pinprick, after general anaesthesia (Figure 28c). The delay in the onset of allodynia in this group of mice could be due to 122 the fact they were still under general anaesthesia in the first few minutes after they were removed from the anaesthetic chamber. All the mice were subjected to a 6-day follow-up period. It was found that 60% and 80% of the mice recovered from the induced allodynia at positions D and P, respectively, on the 6 t h day of the experiment (Figure 28b) 123 First 60 Minutes Isoflurane (2%) ——- Position D o Position P -o —i 120 (b) Figure 28 The effects of general anaesthetics on the development of tourniquet-induced post-ischaemic allodynia in mice (n=5). At t=0, the mice were removed from the anaesthetic chamber. Graph (a) and (b) show the response of the mice to the touch of a piece of VFH in the first 60 minutes of the experiment and in the 6-day follow-up period respectively. Graph (c) shows the pinprick response of another group of normal (control) mice. 6-Day Follow-Up Period Isoflurane (2%) o4 1 1 1 -0 30 60 90 Time (hours) 124 First 60 Minutes Isoflurane (2%) 125 3.4.3. The Effects of Peripheral Administration (Intravenous Injection in the Tail) of Analgesic Agents on the Development of Tourniquet-Induced Post-lschaemic Allodynia in Mice In previous sections, it was found that systemic injections of various analgesic and anaesthetic agents were ineffective in preventing the development of allodynia in mice. In this set of experiment, we investigated the effects of peripheral injections of various analgesic and anaesthetic agents on the development of allodynia in mice. Some of these drugs were able to block pain when administered peripherally, such as the local anaesthetics. This allowed an investigation of the importance of the sensory information transmitted by various peripheral nerve fibres on the development of allodynia in mice. A 20-minute tourniquet was used to induce allodynia in the mouse tails. After the initial screening procedures, the mice were anaesthetized with 2% isoflurane by placing them into a 5L anaesthetic chamber. After the mice had lost their righting and withdrawal reflexes, a 300 mmHg pressure cuff was applied around the base of their tails. This was followed by intravenous injection of one of the test drugs at a site that was close to the tip of the tails. The volume of injection was 0.05 ml. A second tourniquet (plastic tubing) was immediately applied around the tails at a site where the tail diameter was 3.5 mm. After that, the 300 mmHg pressure cuff was released and the mice were immediately removed from the anaesthetic chamber. The duration of the tourniquet was 20 minutes. The presence of allodynia in the tail was, again, assessed by touching the tail with a V F H . The following drugs were tested in this set of experiments: morphine (1.5%), lidocaine (1%), bupivacaine (0.3%), QX-314 (2%), A S A (2.4%) and saline control. In summary, injection of these drugs did not block the 126 development of the tourniquet-induced allodynia; however, the appearance of allodynia in the mouse tails were delayed. In the saline control group (n=5), allodynia at position D was observed in 100% of the mice at 4 minutes after the tourniquets were released. Only 80% of the mice developed allodynia at position P, which was found at 4 minutes after the tourniquets were released. 3.4.3.1.Morphine (1.5%), Lidocaine (1%), Bupivacaine (0.3%) and QX-314 (2%) In the group of mice that received morphine (1.5%, 0.05 ml) (n=5), no allodynia was detected during the first 300 minutes after the tourniquets were released. On the 2 n d day of the experiment, however, 60% of the mice were found to be allodynic at both position D and position P. The percent of mice that were allodynic at positions D and P decreased to 40% on day 4 of the experiment (Figure 29). At the end of the 6-day follow-up period, 40% of the mice in this group were still allodynic at both positions D and P. For the group of mice that received lidocaine (1%, 0.05 ml) (n=5), no allodynia was observed during the tourniquet-period as well as the first 13 minutes after the tourniquet was released. The onset of allodynia detected in this experiment was delayed. Starting at 32 minutes and 22 minutes after the injection, the mice in this group gradually began to respond to touch at position D and position P, respectively, of their tails. 100% of this group of mice were responding positively to touch at positions D and P of their tails at 90 minutes after the tourniquets were released. In the 6-day 127 follow-up period, 40% of the allodynic mice recovered from the induced allodynia at positions D and P on the 2 n d day of the experiment and remained non-allodynic in the rest of the follow-up period. Two mice recovered from the induced allodynia at position P but not D on the 4 t h and 5 t h day respectively. On the 6 t h day of the experiment, the mouse that recovered from allodynia at position P on the 3 r d day of the experiment was found to have recovered from allodynia at position D. For the group of mice that received bupivacaine (0.3%, 0.05 ml) (n=5), no allodynia was detected during the tourniquet period as well as the first 15 minutes after the tourniquet was released (Figure 31a). The onset of allodynia was delayed. The mice in this group gradually began to respond to touch at position D at 30 minutes after the injections. 50 minutes after the injections, allodynia was observed in all the mice in this group. A similar response pattern was observed at position P, but 100% of the mice in this group were found to be allodynic at this position 35 minutes after the injections (Figure 31a). In the 6-day follow-up period (Figure 31b), one mouse was found to have recovered from the induced allodynia at both positions D and P on the 2 n d day of the experiment. On the 3 r d day of the experiment, another mouse recovered from allodynia at both positions D and P and another mouse recovered from allodynia at position P only. On the 4 t h and 5 t h day of the experiment, 60% of mice in this group had recovered from the induced allodynia at both positions D and P. For the group of mice that received QX-314 (2%, 0.05 ml), none of the mice responded to touch at position D on the 1 s t day of the experiment (Figure 32). However, one mouse was found to be allodynic at position P of its tail at 4 minutes after the tourniquet was released. The onset of tourniquet-induced post-ischaemic allodynia at 128 both positions D and P was delayed. On the 2 day of the experiment, all of the mice were found to be allodynic at both positions D and P, but two of these mice only responded to the touch by gently shifting their tails away from the touch stimulus (VFH). On the 3 r d day of the experiment one of the mice had recovered from the induced allodynia at both positions D and P. On the 4 t h day of the experiment, another mouse was found to have recovered from the induced allodynia at position D only. On the 5 t h day, three of the mice were still responding to the touch of a piece of V F H at both positions D and P. Once a mouse recovered from the induced allodynia, it remained recovered in the rest of the 6-day follow-up period. In this experiment, once a mouse recovered from the induced allodynia, it remained non-allodynic in the rest of the follow-up period. On the 6 t h day of the experiment, another mouse was found to have recovered form the induced allodynia at position P only. Also, the recovery of the mice from the induced allodynia was a gradual process. The mice gradually responded to the touch less vigorously until they completely recovered from the induced allodynia. 129 Positions D and P —— Position D O- Position P o 30 60 90 120 Time (hours) Figure 29: The effects of peripheral injection of morphine (0.05ml, 1.5%) on the development of tourniquet-induced post-ischaemic allodynia in mice. Post-ischaemic allodynia was induced by a 20-minute tourniquet (n = 5). A pressure cuff was applied at the base of the tail to occlude circulation to the tail. The drug was injected intravenously into the tail, at t=0 minute, immediately after the pressure cuff was applied. Then, a tourniquet was applied around tail, followed by the release of the pressure cuff. The tourniquet was released at t=15 minutes. 130 First 180 Minutes (Lidocaine 1%, 0.05 ml) 6-Day Follow-Up Period (Lidocaine 1%, 0.05 ml) 0 4 , , • , 0 30 60 90 120 Time (hours) ^ Figure 30: The effects of intravenous injection (distal to the tourniquet-site) of lidocaine (0.05 ml, 1%) into the mouse tails on the development of tourniquet-induced post-ischaemic allodynia at positions D and P of the tails (n=5). The drug was injected at t=0 minute and the tourniquet was released at t=20 minutes. Graphs (a) and (b) show the responses of the mice to touch in the first 180 minutes of the experiment and in the 6-day follow-up period respectively. 131 First 90 Minutes (Bupivacaine 0.3%, 0.05ml) 6-Day Follow-Up Period (Bupivacaine 0.3%, 0.05ml) —•— Position D ---©-- Position P •r • '"O <J"T 1 1 1 1 0 30 60 90 120 Time (hours) ^ Figure 31: The effect of intravenous injection (distal to the tourniquet-site) of bupivacaine (0.05 ml, 0.03%) into the mouse tails on the development of tourniquet-induced post-ischaemic allodynia at positions D and P of the tails (n=5). The drug was injected at t=0 minute and the tourniquet was released at t=20 minutes. Graphs (a) and (b) show the responses of the mice to touch in the first 90 minutes of the experiment and in the 6-day follow-up period respectively. 132 c c O CN tn *- UL <D > O 100 80 60 40-j 20 O +J D) C c o cs tn 2 1 <D > O O 100n First 90-Minutes (QX-314 2%, 0.05 ml) Position D Position P o-ooo-ooo-ooo-ooo«>oo-ooo- o -o o . . . . T . . . . T . . . . T f f——r 0 10 20 30 40 50 60 70 80 90 Time (minutes) 6-Day Follow-up Period (QX-314 2%, 0.05 ml) (a) —— Position D • o - Position P 30 60 90 Time (hours) 120 (b) Figure 32 The effect of intravenous injection (distal to the tourniquet-site) of QX-314 (2%, 0.05 ml) into the mouse tails on the development of tourniquet-induced post-ischaemic allodynia at positions D and P of the tails (n=5). The drug was injected at t=0 minute and the tourniquet was released at t=20 minutes. Graphs (a) and (b) show the responses of the mice to touch in the first 90 minutes of the experiment and in the 6-day follow-up period respectively. 133 3.4.3.2.ASA (2.4%) Intravenous injection (distal to the tourniquet-site) of A S A (2.4%, 0.05 ml) into the tails of a group of mice (n=5) immediately prior to the application of a 20-minute tourniquet was found to be ineffective in preventing the development of post-ischaemic allodynia in their tails. 100% of the tested mice were found to be allodynic at position D within 6 minutes after the release of the tourniquets (Figure 33a). The observed allodynia was not delayed compared to the control group of mice into which saline was injected. The same result was observed at position P (Figure 33b). In the follow-up period, one mouse recovered from the induced allodynia at both positions D and P on the 2 n d day of the experiment. On the 3 r d day of the experiment, 80% of mice responded to touch at position D but only 40% of the mice responded to touch at position P. On the 4 t h day and the 5 t h day of the experiment, another 2 mice were found to be recovered from the induced allodynia at position D. On the 6 t h day of the experiment, 40% and 20% of the mice were found to be responding to touch at positions D and P respectively. 134 First 300 Minutes (Aspirin 2.4%, 0.05 ml) o ra c T3 c O <N Q-»*• W w cu x "~ LL 0) > O O 100-, 80 60-\ 40 20 0 iwaap o » » » » » o 30 60 ~90~ 120 150 Time (min) 6-Day Follow-Up Period (Aspirin 2.4%, 0.05ml) 100 30 60 90 Time (hours) —— Position D --o-- Position P 180 (a) —— Position D -o-- Position P 120 (b) Figure 33: The effects of intravenous injection (distal to the tourniquet-site) of ASA (0.05 ml, 2.4%) into the mouse tails on the development of allodynia at positions (a) D and (b) P of the tails (n = 5). The drug was injected at t=0 minute and the tourniquet was released at t=20 minutes. Graphs (a) and (b) show the responses of the mice to touch in the first 180 minutes of the experiment and in the 6-day follow-up period respectively. 135 3.4.4. The Effects of Lidocaine Nerve Block on the Development of Tourniquet-Induced Post-lschaemic Allodynia in Mice Nerve block (at the base of a mouse tail) was induced by subcutaneous injection of 0.03 ml of 2% lidocaine 10 minutes prior to the application of a 15 minutes tourniquet around the base of the mouse tail. This method was found to be ineffective in blocking the development of post-ischaemic allodynia in all the mice tested (n=5). However, the onset of allodynia was delayed. In these mice, the tail-flick response to touch at position D was absent in all mice in this group until at about 110 minutes after the injection, in other words, during the 15-minute tourniquet period and the first 95 minutes following the release of the tourniquet. But, 100% of the mice were found to be allodynic at position D at about 160 minutes after the injections; 2 mice recovered from the induced allodynia at position D on the 3 r d and 5 t h day of the experiment. The tail-flick response to touch observed at position P was similar to that at position D. Allodynia was not detected at position P until t=110 minutes. The onset of allodynia at this position was delayed. However, only 80% of the mice developed allodynia at this position (at t=180 minutes). Interestingly, all of the 4 allodynic mice recovered completely from allodynia at position P on the 2 n d day of the experiment. 136 First 180 Minutes (Lidocaine 2%, 0.03ml) D) C S c O C N CO ^ *- L L . <D > O O 100 8 0 -6 0 -4 0 -2 0 -0 ffOOOOO 0 o ynnnocoo » » » » 0-0--0--0--0 —— Position D -o-- Position P 30 60 90 120 150 Time (min) 6-Day Follow-Up Period (Lidocaine 2%, 0.03 ml) 180 (a) 100 c c O C M Q- 34-tn * - L i -fl) > o 80H 60H 40-k> 2 0 ^ 04 —— Position D -o-- Position P 30 60 90 Time (hours) 120 (b) Figure 34: The effect of lidocaine (2%, 0.03 ml) induced nerve block on the development of post-ischaemic allodynia in mice (n=5). Lidocaine was injected sc into the base of a mouse tail immediately prior to the application of a tourniquet at t=0 minute. At t=20 minutes, the tourniquet was released. Graphs (a) and (b) show the responses to touch of the mice in the first 180 minutes and in the 6-day follow-up period respectively. 137 4. DISCUSSION 4.1. The Method: Tourniquet-Induced Post-ischaemic Allodynia 4.1.1. Implications from the Time-Course of Tourniquet-Induced Post-ischaemic Allodynia As shown in section 3.1.1, the application of a tourniquet around the base of a mouse tail was found to be effective in inducing allodynia in the tail. The overall duration of the induced allodynia increased as the duration of tourniquet application increased. The percentage of the mice that recovered from the induced allodynia during the 6-day follow-up period decreased with increasing durations of tourniquet application. This could possibly be due to the difference in the severity of tissue damage induced by the ischaemia during the tourniquet period. Longer duration of tourniquet application could have been associated with more severe tissue damage, resulting in a longer duration of the induced allodynia. In order to minimize the pain experience by the mice, unnecessarily long duration of post-ischaemic allodynia should be avoided if possible. With the time course of post-ischaemic allodynia established in the current study, experimenters can choose a duration of tourniquet application that would induce allodynia of sufficient duration with minimal damage to the experimental animals. Interestingly, in spite of a potentially more severe damage associated with a longer duration of tourniquet application, the onset time of the induced allodynia increased with increasing duration of tourniquet application. This could be due to the ischaemic-block of the nerve fibres induced by the application of a tourniquet for more 138 than 20 minutes. After the tourniquets were released, the mice slowly recovered from the ischaemic nerve block, which led to the delay in the onset of the tourniquet-induced post-ischaemic allodynia in the groups where ischaemic nerve block was observed. The recovery periods of the mice that experienced a longer duration of nerve block could be longer than that of those experienced a shorter duration of ischaemic block, resulting in longer delay in the onsets of tourniquet-induced post-ischaemic allodynia in the groups that received long durations of tourniquet application. These results suggested that the firing of the peripheral nerve fibres (pain and/or touch fibres) could be necessary in the appearance of allodynia. Allodynia at position P of a mouse tail developed slower than that at position D. Position P was located proximal to the tourniquet-site, i.e. a non-ischaemic area in the tail, and presumptively undamaged. According to Lewis (1936), the allodynia observed at this position could also be classified as secondary hyperalgesia. A conclusive mechanism (central and/or peripheral) of the development of allodynia at this position could not be determined with the evidence found so far. The delay in the onset of allodynia at position P was more marked for the groups that received longer durations of tourniquet application, which could have resulted from the ischaemic nerve blocks that were associated with long durations of tourniquet application. Allodynia was not developed until the C-fibre signals were restored. Also, once the allodynia was developed, the maintenance of the induced allodynia at position P was independent from the nociceptive signals from the distal part. This will be further discussed in section 4.3.2. The underlying mechanism of the development of allodynia in an undamaged area (secondary hyperalgesia) remains to be investigated. 139 According to Lewis (1936), the hyperalgesia surrounding a faradic injury of the skin was of less extent than that within the injured area. Similar phenomena were observed in our experiments where the percentage of mice that developed allodynia at position P was generally less than that at position D. Arguably, that allodynia developed at all in position P is suggestive of a central rather than peripheral mechanism for its induction. 4 .12 . Strengths and Weaknesses of Tourniquet-Induced Post-lschaemic Allodynia in Mice 4.1.2. l.Strengths Various currently used methods for inducing allodynia and hyperalgesia in experimental animals have been discussed earlier. All those nerve injury methods, which attempt to mimic human conditions of chronic or persistent pain, induce pain that the animal cannot control (Dubner, 1989). It is important for the investigators to assess the level of pain in these animals, and avoid exposing the animals to intolerable pain (Dubner, 1989). As discussed in section 1.1, the method used for inducing allodynia in our study was rapid, convenient, and less invasive to the experimental animals. It was found that most of the mice that received 20 minutes and 30 minutes tourniquet were able to recover completely from the induced allodynia, suggesting the reversibility of the induced allodynia. The induced allodynia was found to be fairly stable on the first day of the experiment, which allowed the evaluation of various analgesic drugs for allodynia. 140 The reversibility of the method we used made it a good alternative to the method for inducing allodynia developed by Kim and Chung (1996). Also, our method for inducing allodynia could avoid excessive tissue damage, and the potential complications resulted from such damages. Potential complications resulted from the surgical procedures performed on the mice could be avoided. Moreover, allodynia was induced with duration of tourniquet application of less than 60 minutes, during which time the mice did not need to be monitored. The procedures were simple and the post-ischaemic allodynia in a mouse tail was induced in a rapid and convenient way. The tourniquet used in earlier experiments performed in our laboratory was a 300mmHg pressure cuff. This type of tourniquet has also been used in other studies with rats (Gelgor, et al., 1986a). However, when this type of tourniquet was inflated around a mouse tail, the plastic balloon (the inflatable part of the pressure cuff) would fold in such a way that incomplete occlusion of blood flow could occur. This could potentially result in a reduction in reproducibility of the allodynia induced with this type of tourniquet. The tourniquets used in all the experiment described in this thesis were made of C-Flex plastic tubing (see section 2.2.2). With this type of tourniquet, the folding observed in the 300mmHg pressure cuff was virtually eliminated. There was virtually no gap observed between the tubing and the mouse tail. Although the pressure might not be evenly applied around the tail because of the potential difference in the thickness around plastic tubing, the gaps between the tourniquet and the skin of the tail was minimized. The reproducibility of the post-ischaemic allodynia induced with this type of tourniquet was found to be improved as compared to that induced by a 300mmHg pressure cuff. 141 Our method for inducing allodynia also minimized the potential injury to the experimental animals. Unnecessarily long duration of tourniquet-induced post-ischaemic allodynia should be avoided if possible. The established time-course of post-ischaemic allodynia can possibly reduce the suffering in mice in future experiments. A minimum duration of tourniquet application can be chosen with respect to the potential action of a test-drug; therefore, the suffering of the experimental animals can be minimized. This method was also found to be effective in distinguishing the central analgesic properties from the peripheral analgesic properties of the few standard analgesic drugs tested in our study. Blockade of the pinprick response was observed in the mice that received morphine systemically but not peripherally, suggesting a central site of action for morphine. Also, the results showed the potential of this method for studying central analgesic properties of various local anaesthetics, such as lidocaine. Analgesic drugs with various modes of actions could be evaluated easily with this method. 4.1.2.2.Weaknesses Even though the tourniquet-induced post-ischaemic allodynia was found to be highly reproducible in the experiment, the variation in the overall duration (the recovery phase) of allodynia was large. For the groups that received tourniquets for more than 20 minutes, the induced allodynia on the first day of the experiment was fairly stable. The small variation in the tourniquet-induced post-ischaemic allodynia observed on the first day of the experiments enabled the evaluation of drugs that target the onset phase of 142 the induced allodynia. However, the variation in the overall duration of the induced allodynia was fairly large, which could limit the sensitivity in the quantification of the actions of analgesic drugs with respect to the recovery phase of the induced allodynia. A fairly large number of mice would be required to establish a more accurate time course for the recovery period of tourniquet-induced post-ischaemic allodynia in mice. Unfortunately, about 5% of the mice failed in the initial screening test. These mice were found to be more sensitive than the others such that they were responding positively to the V F H before a tourniquet was applied. The implication of this observation was that an even larger number of mice were required to be purchased, resulting in an increase in the cost for the experiments. 4.2. Pharmacology 4.2.1. Relationship between analgesic and allodynia blocking doses In Phase II of the study, we determined the doses of the tested analgesic drugs that were effective in blocking the tail-flick response to pinprick in mice. In Phase III of the study, the same analgesics and doses were used again. The effects of these drugs on tourniquet-induced post-ischaemic allodynia in mice were investigated. In general, with a few notable exceptions, the analgesia against pinprick (in Phase II of the study) was paralleled by analgesia against allodynia (in Phase III of the study). 4.2.1.1.Morphine Subcutaneous injection (at the back of the mouse neck) of 10 mg/kg and 20 143 mg/kg morphine was found to be effective in blocking the pinprick pain in normal mice as well as the appearance of allodynia in allodynic mice. 5 mg/kg morphine (sc) was found to be effective in blocking pinprick pain and the appearance of allodynia in only 40% of the mice tested. The durations of blockade of pinprick pain and the appearance of allodynia were very similar for all doses of morphine (Figure 11, Figure 18). This suggested that morphine could have blocked pinprick pain and allodynia in a mouse tail through the same mechanism, presumably a central mechanism. When morphine was injected intraperitoneally with a tourniquet around the base of a mouse tail to prevent the drug from entering the tail, the appearance of allodynia at the mouse tail was also blocked. It is believed that opioid-induced analgesia is due to actions at several sites within the C N S . The observations in these experiments confirm a central acting site for morphine. Moreover, when morphine was injected intravenously into a normal mouse tail with the blood flow to the tail occluded by a tourniquet, the tail-flick response to pinprick was not affected until the tourniquet was released. As the circulation to the tail was restored, the morphine that was previously trapped in the tail by the tourniquet was distributed throughout the body. This allowed the drug to induce analgesia in the mice after reaching its sites of action, which were mainly located in the central nervous system, resulting in the absence of their tail-flick responses. This further confirmed a central rather than a peripheral mechanism for morphine blockade of pinprick pain and the appearance of allodynia. 144 4.2.1.2.Lidocaine Local anaesthetics were used in the experiments for various reasons. Firstly, there exists a large body of literature on systemic lidocaine as a treatment for chronic pain in humans (Dahl, 1993; Wallace et al., 1997). Reduction in intensity or complete relief of chronic pain was reported in human subjects after intravenous injection of low doses of lidocaine (Petersen et al., 1986). Secondly, by blocking the pain and touch fibres locally, the roles of these nerves in the development of tourniquet-induced post-ischaemic allodynia could also be investigated. Small nerve fibres are generally more susceptible to the action of local anaesthetics than the large fibres. Small unmyelinated C fibres, and small myelinated A-8 fibres are blocked before larger myelinated Ay, A(3, and Ace fibres. By selectively blocking the pain fibres with carefully chosen local anaesthetics or their analogues, the role of pain/touch fibres in the development and appearance of post-ischaemic allodynia could be investigated. Subcutaneous and intraperitoneal injections of lidocaine (75 mg/kg) were found to be about equally effective in blocking the pinprick pain in normal mice as well as the appearance of allodynia in allodynic mice. Moreover, the durations of absence of the two different responses were very similar, suggesting lidocaine could have blocked pinprick pain and the appearance of allodynia through the same mechanism. Also, when lidocaine was injected ip with a tourniquet applied around the mouse tail, both pinprick pain and the appearance of allodynia were blocked for similar duration, indicating lidocaine could not have induced analgesia by acting on sites that were located within the tail. The potential central site of action might be located at the spinal or higher level in the central nervous system, which remained to be investigated. 145 On the other hand, when lidocaine (a local anaesthetic) was injected peripherally into the tail that had a tourniquet applied around its base, both pinprick pain and the appearance of allodynia were blocked, confirming its peripheral site of action on the sodium channels on the excitable membranes of various nerve fibres, such as pain and touch fibres. These experiments suggested lidocaine could have blocked the pinprick pain and the appearance of allodynia in mice through two different mechanisms: central and peripheral. Systemic injections of low doses of lidocaine were also found to be effective in relieving chronic pain in human patients, which could be due to the central actions of lidocaine. This is because much greater concentration are needed for a peripheral block than for central analgesia. When lidocaine was injected systemically or peripherally into the mice before the application of a tourniquet, the appearance of allodynia was delayed. After the anaesthetic wore off, the mice responded to the touch by flicking their tails. The injected lidocaine could have masked the appearance of the induced allodynia, but it did not prevent the development of allodynia in the mice. 4.2.1.3.Bupivacaine Bupivacaine (50 mg/kg) was found to be an interesting drug that blocked allodynia but not pinprick pain in mice when injected subcutaneously (at the back of the neck). However, when the drug was injected intraperitoneally, pinprick pain was blocked. Increasing the dose would result in death of the animals; therefore, no higher 146 doses were further tested. Bupivacaine could be more selective for the mechanism involved in the appearance of allodynia. This mechanism may not necessarily involve the perception of pinprick pain. The touch fibres are larger than the pain fibres, which makes them harder to be blocked with local anaesthetics. Bupivacaine could have bound to the N a + channels on the touch fibres more selectively (even at a lower concentration), resulting in the selective blockade of allodynia, given that touch sensation fibres are presumably important in the perception of allodynia. This hypothesis remains to be investigated. 4.2.1.4.QX-222/QX-314 Both QX-222 and QX-314 are positively charged quaternary local anaesthetic analogues with structures similar to that of lidocaine. Their charges make them impermeable through biological membranes and the blood brain barrier. In the experiment, QX-222 was found to be effective in blocking the pinprick pain but not post-ischaemic allodynia in mice when the drug was injected systemically (sc injection at the back of a mouse neck). This suggests that the development and appearance of tourniquet-induced post-ischaemic allodynia in mice could have involved other sensory mechanisms, such as the touch fibres, that were not affected by the injected QX-222. On the other hand, peripheral injection of QX-314 (iv injection into the mouse tails) was found to be effective in only delaying the development of tourniquet-induced post-ischaemic allodynia in mice. Although the pinprick pain of all the mice that received QX-314 (iv injection into the mouse tails) (n=5) was blocked and none of these 147 mice were found to be allodynic on the 1 day of the experiment, all of them were found to be allodynic on the 2 n d day of the experiment. The appearance of allodynia after the prolonged local anaesthesia induced by QX-314 suggested that the development of tourniquet-induced post-ischaemic allodynia in mice might not require the signals that were sent to the C N S from the peripheral pain fibres. Nerve sensitization process might have taken place at the peripheral nerve fibres, resulting in allodynia in the tails. The injected QX-314 only masked the appearance of the induced allodynia, but it did not affect the development of allodynia in the mice. It has been shown that large doses of lidocaine could induce irreversible damages to nerve fibres (Bainton and Strichartz, 1994). In these cases, the blockade of the nerve fibres induced by lidocaine was irreversible. Although the potential damage induced by large doses of QX-314 is currently unknown, since QX-314 is a local anaesthetic analogue and it possesses some pharmacological properties of tertiary local anaesthetics, it is not unreasonable to assume that high doses of QX-314 can also induce damages to the nerve fibres. In the experiment, the appearance of allodynia in a group of mice that received 2% QX-314 peripherally prior to the tourniquet application was delayed for about 1 day. A 20-minute tourniquet was employed for inducing allodynia in these mice. Although some of the mice responded to touch by flicking their tails gently on the 2 n d day of the experiment, 100% of the mice were found to be allodynic on this day, which was not evident in mice that received other tested drugs. The dose of QX-314 used in this experiment was fairly high, which might have induced additional tissue damages to that induced by the ischaemia during the tourniquet-period. However, compared to the groups of mice that received other local 148 anaesthetics, the recovery rate of this group of mice (received QX-314) from the induced allodynia was found to be similar. This group of mice also responded to pinprick and touch by flicking their tails on the 2 n d day of the experiment, suggesting that the blockade of the nerves induced by the injected QX-314 was not irreversible. Therefore, we believe that damages to the peripheral nerves in the mouse tails, that was induced by the injected QX-314, was minimal. 4.2.1.5.Pentobarbital According to Hobbs et al. (1996), pentobarbital belongs to the barbiturate class of general depressant. "It reversibly depresses the activity of all excitable tissues, and it can produce all degrees of depression of the C N S , ranging from mild sedation to general anaesthesia. However, its direct actions on peripheral excitable tissues are weak. Pain perception and reaction are relatively unimpaired until the moment of unconsciousness". (Hobbs etal . , 1996) Intraperitoneal injections of pentobarbital (30 mg/kg, 40 mg/kg) had no effect on the tail flick responses of normal mice to pinprick. The mice were sedated and their righting reflexes were lost, but their pinprick responses of were not affected. On the other hand, the tail-flick responses of allodynic mice were briefly blocked by 40 mg/kg but not 30 mg/kg pentobarbital. In the experiment, all the mice were sedated and their righting reflexes were lost for a much longer period than the duration of the blockade of the appearance of allodynia. In the experiment, the pinprick pain perceived by the mice that received 149 pentobarbital intraperitoneally was not affected. Systemic injections of pentobarbital, lidocaine and morphine induced similar decline in performance of the mice on a rotarod. Only the mice that received pentobarbital (ip) lost their righting reflexes. This was not evident in the mice that received either lidocaine (ip and sc) or morphine (ip and sc). This piece of experimental evidence suggested that lidocaine, like morphine, could possess central analgesic properties. The blockade of pinprick pain and the appearance of allodynia induced by systemic injections of lidocaine and morphine could possibly be due to their specific central actions rather than simply depressing the C N S . 4.2.1.6.Rocuronium Some of the tested drugs, such as the local anaesthetics and their analogues, could possibly block the sodium channels on motor fibres. This could potentially block the ability of a mouse to withdraw its tail from a painful stimulus. Therefore, the rotarod was employed to examine the motor effects of the injected analgesics on mice. Also, the abilities of the mice to walk on the experiment bench and their spontaneous tail movements were monitored. In order to show pharmacologically that the tested compound blocked the tail-flick response of a mouse by blocking the perception of pain rather than by blocking their ability to respond to the stimulus, rocuronium was included in the study. Intraperitoneal injection of a very high dose of rocuronium (0.7 mg/kg) was found to be capable of reducing the performance of the mice on a rotarod but ineffective in blocking their tail-flick responses to pinprick. Compared to the mice that received rocuronium, it was very unlikely that the motor function of the mice received 150 other tested drugs were affected. 4.2.2. How do the effects of the analgesic agents on prevention, development, and duration of tourniquet-induced post-ischaemic allodynia compare? Systemic injections of morphine (10 mg/kg, 20 mg/kg), and the tested local anaesthetics (except bupivacaine) into normal mice were found to be effective in relieving the pinprick pain, indicated by the absence of their pinprick responses after the injections. These drugs (at the same doses, except QX-222) were also found to be effective in blocking the appearance of allodynia in the allodynic mice when injected through the same routes of administration (systemic injection). However, pre-treating the mice with these drugs (systemic administration) was found not to block the development of post-ischaemic allodynia induced by a 20-minute or 30-minute tourniquet. Generally anaesthetizing the mice was also found to be ineffective in blocking the development of tourniquet-induced post-ischaemic allodynia. However, all these drugs delayed the appearance of allodynia to various extents. This suggested a complex mechanism for the development of post-ischaemic allodynia in mice where conscious perception of pain might not be the only or an essential contributing factor to the development. Intravenous local injection of various local anaesthetics into a mouse tail prior to the application of a tourniquet was also ineffective in blocking the development of post-ischaemic allodynia. Firing of the pain fibres was presumably blocked by the local anaesthetics, as indicated by the negative response to the pinprick test at the tails after the drugs were injected locally into the mouse tails. All the nociceptive information 151 transmitted by the pain fibres (A-8 and C-fibres) and the touch information transmitted by the A-p fibres should have been blocked by the local anaesthetics. However, allodynia still developed in the mouse tail, albeit with a delayed onset. This suggests that a peripheral mechanism that was localized in the tail could be involved in the development of post-ischaemic allodynia. In another experiment, lidocaine (2%) was injected to produce nerve block into the mouse tails at sites that were proximal to the tourniquet-sites. The nerve block induced in the mouse tails was confirmed by the negative responses to the pinprick test at various positions on the tails after the drug was injected. This method was found to be ineffective in blocking the development of post-ischaemic allodynia at position D of the mouse tails, but its appearance was delayed, suggesting that the development of allodynia could be independent from the transmission of the nociceptive information to the central nervous system though the pain fibres. In other words, a peripheral mechanism of development of post-ischaemic allodynia could be involved. However, although allodynia was detected at position P in 60% of the mice on the first day of the experiment, none of these mice were found to be allodynic during the 6-day follow-up period. This suggested that the maintenance of allodynia in an undamaged area could be dependent on some sensitized neurons located in the C N S , otherwise, the proximal nerve block would not have sped up the recovery of the mice from the induced allodynia. The transient allodynia observed on the first day of the experiment could be due to (1) the spread of the algesic substances released from the injured tissue as part of the acute inflammatory response, and/or (2) the activation of the chemosensitive afferents by these products of inflammation. LaMotte et al. (1991) proposed that the 152 chemosensitive afferent fibres could be widely branched and could terminate in the dorsal horn on interneurons that receive input from cutaneous mechanoreceptors. What could have happened in the tail during the tourniquet period? The ischaemic condition might have led to the build-up of various products of inflammation in the mouse tails. Even though injection of A S A was found to be effective in preventing the development of reperfusion hyperalgesia in rats (Gelgor et al., 1986b; Gelgor et al., 1992b), it was found to be ineffective in preventing the development of post-ischaemic allodynia in the mice in our study. In our study, neither systemic injection of various doses of A S A (100 mg/kg, 300 mg/kg, and 400 mg/kg) nor peripheral injection (iv injection into the tails) of A S A (2.4%) prior to the application of a tourniquet was effective in preventing the development of post-ischaemic allodynia in mice. Moreover, the overall time course of allodynia of the group that was pre-treated with A S A , which should have inhibited the production of prostaglandins (one of the major products of inflammation), was not significantly (P>0.05) different from that of the saline control group. This suggested that either the build-up of prostaglandins in the tail during the ischaemic period was not important in the development of post-ischaemic allodynia, or the build-up of prostaglandins in the tail during the ischaemic period was not the only contributing factor to the development of post-ischaemic allodynia. 153 4.3. Implications 4.3.7. Animal Care "The nerve injury methods which attempt to mimic human conditions of chronic or persistent pain induce pain that the animal cannot control" (Dubner, 1989). Pain experienced by the experimental animals should be minimized. As discussed earlier, there are a few methods for inducing allodynia in experimental animals; however, these methods normally involve the injection of toxic chemicals, such as capsaicin (Simone et al., 1989; Torebjork et al., 1992; Sang et al., 1996; Kupers et al., 1997), nociceptin (Hara et al, 1997; Calo et al., 1998), mustard oil (allylisothiocyanate) (Cervero and Laird, 1996) and formalin, or the use of irreversible surgical procedures (Kim and Chung, 1991). As an alternative to time consuming and invasive procedures, and the necessity of producing excessive tissue damage, a tourniquet made of a piece of plastic tubing was applied onto the tail of a mouse for the induction of allodynia. The results from the current study suggested that the current method would be a good alternative method to those listed above in terms of speed and convenience. 4.3.2. Mechanism of Tourniquet-Induced Post-lschaemic Allodynia 4.3.2.1.Central Site: Effect of Isoflurane and Morphine We investigated the role of the perception of pain at the C N S level in the development of allodynia. Both isoflurane and morphine were found to be ineffective in blocking the development of tourniquet-induced post-ischaemic allodynia. However, they were both effective in blocking the appearance of post-ischaemic allodynia. This 154 showed that the conscious perception of pain might not be the only contributing factor in the development of tourniquet-induced post-ischaemic allodynia. Various factors and products of inflammation had been shown effective in decreasing peripheral pain sensation threshold (Reeh et al., 1997). The built-up products of inflammation within the tails during the tourniquet-period could have sensitized the peripheral nerve fibres, resulting in the development of tourniquet-induced post-ischaemic allodynia in the tails. Although the development of post-ischaemic allodynia in the mice in our experiments could possibly be due to the sensitization of the peripheral nerve fibres within the ischaemic area in the mouse tails, the maintenance of the induced allodynia in the tails could have involved various sensitization processes in the CNS. As the analgesic and anaesthetic drugs wore off, the perception of pain in the tails returned. At this time, the nociceptive signals transmitted through the primary nociceptive afferents could have induced various sensitization processes in the CNS, which contributed to the maintenance rather than the development of post-ischaemic allodynia in the tails. We have shown that peripheral injections of various local anaesthetics were only able to block the appearance of allodynia at position D but not P. In this case, the appearance of allodynia at position P was not dependent on the appearance of allodynia at position D, suggesting that a central sensitization process could have occurred. Once it happened, the appearance of allodynia at position P was maintained by the C N S ; consequently, the blockade of the appearance of allodynia at position D induced by local anaesthetics did not affect the appearance of allodynia at position P. Kaneko et al. (1997) have shown that the responses of rat's dorsal horn neurons to touch sensation transmitted from the A-p fibres could be enhanced by spinally 155 administered G A B A A receptor antagonists. On the other hand, Ito et al (1997) have shown that intrathecal injection of MK-801, an NMDA receptor antagonist, could inhibit PGE 2 - induced allodynia in mice. The G A B A and NMDA receptors could be involved in the development and maintenance of tourniquet-induced post-ischaemic allodynia in mice in our study. The functional role of these two types of receptors in the development and maintenance of tourniquet-induced post-ischaemic allodynia in mice has still to be determined. In our experiment, we have shown that systemic injection of morphine was unable to block the development of allodynia, and the delay of allodynia induced by morphine was reversible by naloxone. Also, the appearance of allodynia at both position D and P was observed within 5 minutes after naloxone was injected. We believed that allodynia had already been developed at both positions before the injection of naloxone. The injected morphine should have blocked the release of the neurotransmitter from these primary nociceptive terminals. Therefore, it was unlikely that the sensitization process occurred at the synapse between the dorsal horn neurons and the terminals of the primary afferents. The central sensitization process could have happened at higher levels at the C N S , and the activation was independent from the signals transmitted by the nociceptive pathways that were affected by morphine. 4.3.2.2.Peripheral: Lidocaine, Bupivacaine, and QX314 Although the maintenance of allodynia in mice might not require the input from the peripheral nerve fibres, the initiation of the sensitization process could possibly require the signals transmitted by these fibres. Lidocaine and bupivacaine are local 156 anaesthetics, which reversibly block the N a + channels on the membranes. QX-314, a local anaesthetic analogue, also possesses similar pharmacological properties as lidocaine and bupivacaine. When injected peripherally, these drugs blocked the pain associated with the application of the tourniquet, which could be a result of the pressure induced by the tourniquet or products of inflammation produced during the period of ischaemia. However, post-ischaemic allodynia still developed in the tails even with the pain, induced by the application of the tourniquet, blocked with the peripheral administration of the local anaesthetics. This suggested that tourniquet-induced post-ischaemic allodynia could have developed through a peripheral mechanism, such that the peripheral nerve fibres were sensitized directly during the ischaemic period in the tails. Various products of inflammation could have been produced in the ischaemic areas in the mouse tails. In section 1.4.2.1, I have already mentioned a list, described by Reeh et al. (1997), of compounds that have been shown effective in reducing the paw withdrawal threshold in rats when administrated peripherally. These substances could have contributed to the development of post-ischaemic allodynia in mice in our experiments through various mechanisms of actions. They might have activated and/or directly sensitized various peripheral nerve fibres. As proposed by Schmidt et al. (1994), the nerve fibres may possess various "sleeping receptors" which can be recruited during inflammation, resulting in a reduction of firing thresholds of the nerves involved. The products of inflammation that were produced in the ischaemic areas in the mouse tails could have activated these "sleeping receptors" and reduced the firing thresholds of the pain and/or touch fibres innervated the ischaemic areas, resulting in the allodynia observed. All these process could have activated various sensitization 157 processes in the C N S , which further contributed to the maintenance of allodynia in mice. Interestingly, nerve block by lidocaine delayed but did not block the development of allodynia either at position D or P on the 1 s t day of the experiment. All mice recovered from allodynia at position P, but not D, on the second day of the experiment. Therefore, the development of allodynia in a non-ischaemic area could potentially be blocked. The nerve block induced by lidocaine could have reduced the magnitude of sensitization in the peripheral nerves, resulting in shortened duration of allodynia observed at position P in the mice. A peripheral mechanism could be involved in the development of allodynia (secondary hyperalgesia) at position P, a claim that is also supported by experimental findings of Lewis (1936) and LaMotte (1991). This reduction in the duration of the induced allodynia was not evident in other experiments discussed, which could possibly be due to insufficient blockade induced by systemic injections of local anaesthetics on the peripheral nerve fibres. According to Lewis (1936), "nocifensor" nerves could be involved in the spread of secondary hyperalgesia into undamaged parts surrounding the damaged area of the skin. These fibres were arranged as arborisations of axons in the skin, and that belonged neither to the sensory nor to the sympathetic system. On the other hand, LaMotte (1991) suggested that there could be widely branched chemosensitive afferent fibres which mediated the spread of hyperalgesia into the undamaged areas of the skin. Both of these hypotheses could support a claim that a peripheral mechanism may be involved in the transient spread of allodynia to position P. Some algesic products of inflammation could have activated the nocifensor neurons or the widely branched chemosensitive afferent fibres, resulting in 158 the allodynia observed at position P. Also, as discussed before, peripheral activation of the nociceptive fibres that innervated the non-ischaemic area could have initiated a central sensitization process, which further contributed to the maintenance of the allodynia in the mouse tails. Since the allodynia induced at position P was not as consistent as that observed at other positions on the tails, a larger sample size would be necessary to confirm the effects of various analgesic/anaesthetic agents on the development of allodynia at position P in mice. The study of the allodynia induced at position P can also be improved by applying the tourniquet at a site that is closer to the middle of the tail, resulting in a larger area for investigation of the development of allodynia at this non-ischaemic part of the tail. 4.4. Complications 4.4.1. Can this model be applied to other species? Post-ischaemic allodynia has also been reported on other animals, such as rats (Gelgor, et al., 1986a). Moreover, a tourniquet is often used in various surgical procedures in humans to occlude blood flow towards distal extremities. The patients who undergo these types of surgeries often report post-surgical chronic pain at distal extremities. The advantages of using mice had been discussed earlier. However, with a larger animal (such as a rat), the blood vessels in the tail could be cannulated, which allows the conditions in the tail to be controlled by the experimenter. Drugs or other chemicals can be constantly modified according to the interests of the investigators 159 during the tourniquet-period. This can possibly lead to a better understanding about the mechanism of post-ischaemic allodynia, allowing the development of better analgesic for the symptom. 4.4.2. Central acting sites for lidocaine and other local anaesthetics Lidocaine was found to be effective in inducing analgesia in both normal and allodynic mice when it was injected systemically into the mice when the blood flow to their tails was occluded by tourniquets. The blockade of pinprick pain and the blockade of the appearance of allodynia in mice that received lidocaine systemically were found to be similar to that induced by systemic injection of morphine. Resemblances between the analgesic effects induced by these two drugs have also been found in other studies. According to Woolf and Wiesenfeld-Hallin (1985), "both lidocaine and narcotic analgesics decrease C-fibre evoked activity in the spinal cord with minimal effects on A-fibre input, and both modify clinical pain without reducing the sensory effects of pinprick or pinch". They also proposed that these two different classes of drugs might share a common post-synaptic mechanism which is activated following their binding to separate sets of receptors (Woolf et al., 1985). It has been shown that opiates reduced the depolarizing action of glutamate by impairing N a + channels and that procaine has a very similar action which is not naloxone reversible (Woolf et al., 1985). A piece of supporting evidence from our study was that the analgesia induced by systemic injections of lidocaine was also not reversed by naloxone. Although the pinprick pain perceived by the mice was blocked in our experiment, which was different from findings by Woolf and Wiesenfeld-Hallin (1985), we should be aware that the 160 doses which we employed in our experiments were fairly high. Therefore, even though some our results were different from what proposed by Woolf et al., we demonstrated that lidocaine could induce analgesia in mice by acting on sites that are not located in the mouse tails. The analgesia induced by systemic injection of lidocaine could have involved the mechanism described by Woolf et al (1985). On the other hand, according to Schwarz and Puil (1998, 1999), the analgesia induced by systemic injection of lidocaine could be due to its effects on the thalamocortical neurons. They have shown that lidocaine at low concentration produces membrane shunting effects on rats' ventral posterolateral nucleus of the thalamus, which were not mediated by the G A B A A receptors. However, the central mechanisms of actions of lidocaine and other local anaesthetics remain to be elucidated. 4.5. Conclusion 4.5.1. Mechanism of Post-ischaemic Allodynia The application of a tourniquet around the base of a mouse tail was found to be effective in inducing post-ischaemic allodynia in the tail. Moreover, it was also found to be convenient for studying the mechanism of and treatment for tourniquet-induced post-ischaemic allodynia in mice. Even though the exact mechanism of the development of post-ischaemic allodynia is still unknown, we have obtained some important information. The appearance of post-ischaemic allodynia could be blocked by drugs that possessed central and/or peripheral analgesic properties, such as morphine, lidocaine, and general anaesthetics. However, the administration of these drugs could only block or delay the 161 appearance of post-ischaemic allodynia. Once the allodynia was developed, the analgesic drugs tested were ineffective in curing the allodynic animals. The recovery phase of tourniquet-induced post-ischaemic allodynia was not shortened by any of the tested analgesic agents in the 6-day follow-up period. None of the analgesic/anaesthetic drugs tested so far was found to be effective in blocking the development of post-ischaemic allodynia. Both central and peripheral mechanisms could be involved. Neither blocking the conscious perception of pain systemically nor the nerve impulses peripherally was effective in blocking the development of post-ischaemic allodynia. Peripherally, the build-up of products of inflammation could have sensitised the nerve fibres and nerve endings, resulting in allodynia. Centrally, sensitisation process could have taken place at various sites, such as in the spinal cord or at higher levels in the central nervous system. The central sensitization could be induced by the peripheral nociceptive signals transmitted through various nociceptive primary afferents. The injections of various analgesic and anaesthetic drugs could block the perception of pain but not the sensitization processes that could have occurred at the nerve fibres innervating the ischaemic areas in the mouse tails. As the analgesic and anaesthetic agents wore off, the perception of pain in the mouse tail returned. The nociceptive information transmitted from the allodynic tails through various pain fibres to the C N S could then have induced changes in the C N S , which further contributed to the maintenance of the induced allodynia in the mouse tails. This could possibly explain the phenomenon that none of the analgesic and anaesthetic agents could block the development of tourniquet-induced post-ischaemic allodynia in the mouse tails. 162 4.5.2. Potential of the Method The application of a tourniquet around the base of a mouse tail was found to be effective in inducing post-ischaemic allodynia in the tail, especially in the parts that were distal to the tourniquet-site. Moreover, one of the advantages of using this method was that the systemic circulation was technically isolated from the tail; therefore, the potential site of action (systemic vs. peripheral) of a drug for pain/allodynia could possibly be located more conveniently. Furthermore, by carefully changing the chemical composition of the fluid in the tail, the effects of each product of inflammation mentioned in section 1.4.2.1 on the development and the duration of allodynia could possibly be studied in detail. As mentioned in the introduction section, NMDA receptors (Ito et al., 1997) and G A B A receptors (Kaneko et al., 1997) could have played an important role in the development of allodynia. These receptors are also correlated with the overall function of the central nervous system. Long-term potentiation (LTP) can be defined as the enhancement of synaptic transmission that occurs at various C N S synapses following a short (conditioning) burst of pre-synaptic stimulation (Rang et al., 1996). NMDA receptors had been shown to play an important role in the process of LTP, because the NMDA antagonists were found to be effective in preventing LTP. Even though the relationship among LTP, learning and memory is still controversial, NMDA-receptor antagonists applied to the hippocampus has been found to be effective in impairing the ability of rats to learn a maze. According to Ito et al. (1997), the injection of MK-801, an NMDA-receptor antagonist, was found to be effective in blocking P G E 2 induced allodynia in mice. If NMDA receptors are involved in the development/maintenance of 163 tourniquet-induced post-ischaemic allodynia, our method of inducing allodynia could potentially be applied to studies of learning and memory, provided that the peripheral contribution to the development of tourniquet-induced post-ischaemic allodynia can be blocked or minimized. Also, drugs for improving memory and learning ability could potentially be developed. 4.5.3. Future A venues for Research The effects of lidocaine (or other local anaesthetics) induced spinal blockade on tourniquet-induced post-ischaemic allodynia have not been investigated yet; therefore it could be a potential future focus of our study. Even though the exact mechanism of tourniquet-induced post-ischaemic allodynia is still unknown, the possibility of a central mechanism has not yet been ruled out. By inducing spinal blockade in a mouse, the signals transmitted from the primary afferent would be blocked at the spinal level in the C N S . If the development of post-ischaemic allodynia was blocked, the central site for the development of allodynia might be located in the spinal cord. However, if the development of post-ischaemic allodynia was not blocked, a peripheral sensitisation mechanism and/or a central sensitisation mechanism located at a higher level in the C N S might be involved. In the latter case, the information carried to the brain from the primary afferent of various nerve fibres probably would not be carried through the nerves that were blocked by the spinal nerve block. Moreover, the results obtained in the current study suggested that both peripheral and central mechanisms could be involved in the development of post-ischaemic allodynia. Complex interactions among various events happening during the 164 ischaemic period in the tail could have contributed to the development of allodynia to different degrees. Peripheral tissue damages induced by the ischaemia could have initiated the development of post-ischaemic allodynia in the mouse tails. On the other hand, the central sensitization processes, which were initiated by the peripheral nociceptive inputs, could have contributed to the maintenance of the induced allodynia in the mouse tails. A combination of the drugs that could block pinprick pain and the appearance of allodynia may be effective in blocking the development of post-ischaemic allodynia. Despite the difficulties in determining the right combination of drugs, this proposal can potentially determine the important components in the development of post-ischaemic allodynia as well as underlying mechanism. 165 5. Bibliography Bainton.C.R. & Strichartz.G.R. (1994) Concentration dependence of lidocaine-induced irreversible conduction loss in frog nerve. Anesthesiology, 81, 657-667. Bellissimo.A. & Tunks.E. (1984) Dimensions of Pain. 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