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The effect of diabetes mellitus on nervous tissue Rivers, William Alexander 1951

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l£3  3?  THE EFFECT OF DIABETES MELLITUS ON NERVOUS TISSUE  by  WILLIAM ALEXANDER RIVERS  k THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MISTER OF ARTS  i n the Department of BIOLOGY AND  BOTANY  life accept t h i s thesis as conforming t o the standard required from candidates  f o r the  Degree of MASTER OF ARTS  Members of the Department of Biology  The  University of B r i t i s h Columbia A p r i l , 1951  Abstract  A, group of alloxanized rats fed on a high s a l t d i e t were studied h i s t o l o g i c a l l y to determine the d i r e c t or i n d i r e c t e f f e c t of diabetes mellitus on the nervous systems  The most s i g n i f i c a n t  changes observed were i n the vascular system of the brain*  Lesions  of the a r t e r i e s were i d e n t i c a l with those found i n a r t e r i o s c l e r o s i s and massive hemorrhages accompanied by multiple d i f f u s e diapedesis not only i n the a r t e r i e s but a l s o i n the veins was hypertension*  i n d i c a t i v e of  I t i s believed the hypertensive condition was  induced  by a combination of the diabetes and s a l t feeding. Associated 'with the vascular dysfunction generalized cerebral changes were noted.  They included neuron  degeneration,  exhibited by l o s s of N i s s l granules^ vacuolization of the and cerebral softening.  cytoplasm  Also Gitter c e l l invasion and p r o l i f e r a t i o n  occurred at the s i t e s of vascular disturbance.  I t appears that the  neurological involvement i n diabetes i s a d i r e c t r e s u l t of changes i n the blood system and not of the disease i t s e l f .  ii  Acknowledgements  The author wishes to express h i s thanks to Dr. A.H. Hutchinson, under whose d i r e c t i o n t h i s research was c a r r i e d out. A. p a r t i c u l a r debt of gratitude i s owing f o r the invaluable fundamental h i s t o l o g i c a l methods and procedures I have learned i n our association as professor and student. I am indebted a l s o , to Dr. W.C.  Gibson who suggested  the problem and was responsible f o r obtaining the experimental material.  Without Dr. Gibson's patient supervision, the s i l v e r  techniques f o r nervous t i s s u e would never have succeeded. To Dr. J.A» Allardyce, f o r h i s kind advice and h e l p f u l suggestions, I am most g r a t e f u l .  His teachings i n physiology have  done much to elucidate the problem i n t h i s aspect* thanks go to Dr. A»L. Chute, who supplied the experimental material as a supplementary problem i n conjunction with h i s own work on diabetes and hypertension.  iii  Table of Contents Page Abstract  . . . . . . . . . .  Acknowledgements Table of Contents  •  i i  • .  i i i  Introduction . . . . . . . H i s t o r i c a l Review  . . . . . . . .•  Diabetic Neuropathy  1 2  . . . . . . . . . . . .  3  .•.  7  •»  11  Alloxan Diabetes  Materials and Methods  i  . • •  Results and Observations  15  Gross. Examination H i s t o l o g i c a l Examination  15 . . . . .  16  Discussion * . « . « • • • » . . . . . . . . . . .  20  Summary  26  • . . . . . . . . . . . . . . . . » • • •  Conclusions Bibliography  . . . . . . . . . . . . . . . . . . . .......  27 28  Introduction  The nervous tissue of alloxanized r a t s was studied to determine the d i r e c t or i n d i r e c t e f f e c t of diabetes on the c e n t r a l nervous system. The diabetic syndrome i s p r i m a r i l y a disturbance carbohydrate metabolism*  Diabetes mellitus such as occurs i n humans, i s  s t i l l a c l i n i c a l syndrome of unknown etiology^ system complications  i n the regulation of  The c e n t r a l nervous  of diabetes include involvement of the p e r i p h e r a l  nerves, s p i n a l cord, b r a i n and autonomic nervous system, as w e l l as those pathologic changes •which develop i n association with the hypoglycemic state•  A r t e r i o s c l e r o s i s i s an e x t r a o r d i n a r i l y frequent accompaniment  of diabetes and i t may w e l l be that many of the above-^nentioned complications,  as w e l l as syndromes of both f o c a l and d i f f u s e cerebral  involvement may develop secondary to disease of the cerebral and meningeal arteries«  I t i s the purpose of t h i s problem t o f i r s t investigate the  vascular system of the b r a i n using general and s p e c i f i c s t a i n i n g procedures and then t o examine the nervous tissue f o r pathologic changes by means of s p e c i f i c s i l v e r staining methodso  A-preliminary  research  by Dr. AoL» Chute has indicated the presence of a r t e r i o s c l e r o s i s as w e l l as a hypertensive condition i n other parts of the experimental animals including the kidney, l i v e r and pancreas*  1  H i s t o r i c a l Review  The cause of diabetes mellitus i s unknown.  The r e l a t i o n s h i p  between diabetes and a r t e r i o s c l e r o s i s , which frequently are found together, i s obscure.  The e f f e c t of diabetes on the nervous system i s as  yet u n s a t i s f a c t o r i l y explained. I t i s w e l l known that nervous system complications may  occur  i n association with diabetes m e l l i t u s . Certain of these coexisting neurologic manifestations appear frequently, and they are e a s i l y recognized from a c l i n i c a l point of view.  Others are l e s s often  encountered and consequently many observers may not i n t e r p r e t them as nervous system complications of t h i s disease. Often i t i s stated that the d i a b e t i c process has a s p e c i a l p r e d i l e c t i o n f o r the peripheral nerves* and those changes associated with peripheral nerve dysfunction have been most widely described (DeJong, 1950).  The pathologic process may not, however, be l i m i t e d to  the p e r i p h e r a l portions of the nervous system, and involvement of the s p i n a l cord, b r a i n stem, midbrain and cerebrum, as w e l l as the autonomic nervous system, has been observed.  In f a c t , there may be a d i f f u s e  a f f e c t i o n of the entire nervous system, i n c l u d i n g both the c e n t r a l and peripheral portions.  The part played by vascular changes, e s p e c i a l l y  cerebrovascular disease, i n the development of neurologic  manifestations  has not been stressed. The cerebral complications of diabetes have never been f u l l y described.  Casamajor and Geipel noted glycogen droplets i n the p e r i -  vascular lymph spaces of the b r a i n and i n the pyramidal c e l l s .  Warren  3 (1938) has seen glycogen droplets i n the g l i a l c e l l s i n a few cases, but does not consider them s i g n i f i c a n t .  White described d i l a t i o n of the  cerebral vessels, thickening of the arachnoid, f o c i of edema with lymphocytic i n f i l t r a t i o n s , g l i a l p r o l i f e r a t i o n , and displacement of the ependyma.  I t must be r e c a l l e d , however, that these postmortem changes  must be d i f f e r e n t i a t e d from those a l t e r a t i o n s of structure which may  be  the r e s u l t of vascular damage, hypoglycemia and i n f e c t i o n . The more obvious carbohydrate disturbance of what i s commonly termed diabetes mellitus has overshadowed the generalized nature of the disease, the etiology of which i s s t i l l unknown.  The concept of  vascular damage as a complication of diabetes must be amended i n the l i g h t of current observation*  I t i s to be recognized rather as an  i n t e g r a l manifestation of the basic disorder, an associated concomitant of the disease.  Such a b i o l o g i c a l approach to t h i s problem was  postu-  lated by Dry and Hines (1941) on the basis of "an inherent weakness which a f f e c t s both the insulin-producing tissues and the vascular system" with a common mechanism f o r the production of a r t e r i o s c l e r o s i s , r e t i n o pathy and neuropathy on the basis of involvement of the nutrient vessels of these structures.  The diabetic animal provides i d e a l material f o r the  study of the phenomena of aging and associated degenerative diseases  since  i t has been demonstrated that diabetes represents an acceleration of these processes.  Diabetic Neuropathy Diabetic neuropathy, or diabetic n e u r i t i s , i s the neurological complication most frequently associated with diabetes m e l l i t u s . f i r s t described i n 1864 by C a l v i who  I t was  observed pains i n the d i s t r i b u t i o n  of the s c i a t i c nerves along with areas of anesthesia i n diabetes.  4 He suggested that the neurologic lesions were the r e s u l t rather than the cause of the disorder*  Since.thenj there have been many c l i n i c a l des-  c r i p t i o n s of the peripheral nerve changes that may The manifestations  of diabetic neuropathy are i d e n t i c a l with  other types of peripheral n e u r i t i s * pareses and ataxia.  occur i n diabetes•  There i s pain, paresthesias, f l a c c i d  Also disturbances  of cutaneous and  sensations, r e f l e x changes and l o s s of motor power.  proprioceptive  There have been  several attempts to c l a s s i f y the peripheral neuropathy associated with diabetes m e l l i t u s . 1.  Jordan (1936) has suggested the following categories:  The hyperglycemic type i s characterized mainly by tenderness of the nerves.  2.  The c i r c u l a t o r y type i s manifested by symptoms such as p a i n and paresthesis and signs such as hyporeflexia and  3«  hyperesthesia.  The degenerative type begins i n s i d i o u s l y and progresses slowly over a period of years.  4.  The n e u r i t i c type, which i s the commonly described diabetic n e u r i t i s , i s marked by pain and paresthesias and there i s weakness which i n some cases progresses to p a r a l y s i s * Treusch (1945) has proposed a s i m i l a r c l a s s i f i c a t i o n -with an  a d d i t i o n a l d i v i s i o n , "diabetic v i s c e r a l n e u r i t i s } which includes the 11  cases of autonomic nervous system involvement.  Patients have a burning  paresthesias of the feet and u s u a l l y show vasomotor changes i n the extremities. The mechanism,' of peripheral neuropathy i n diabetes i s not known.  I t may  develop i n association with any degree or stage of the  disease, and occasionally i t i s the f i r s t symptom. older patients but not uncommon i n the young.  I t i s more common i n  The pathological changes  5 are generally a demyelinization of peripheral nerves« Harris (1922) has indicated the close s i m i l a r i t y with b e r i b e r i and so-called a l c o h o l i c n e u r i t i s and suggested a vitamin B d e f i c i e n c y could be responsible.  However, i t has been found that the neuropathy  may develop i n patients who  have been on an adequate d i e t . (Needles,  1930). Atherosclerosis has been c i t e d by various workers as of poss i b l e s i g n i f i c a n c e i n the development of dysfunction of the p e r i p h e r a l nerves, due to the resultant ischemia and anoxia of the nerves. However, once again, diabetic neuropathy may show no apparent peripheral a r t e r i o s c l e r o s i s .  occur i n young persons  who  I t i s probable that neither  tMamine d e f i c i e n c y nor vascular disease i s alone responsible f o r the nervous iiupset and i t may be that the peripheral nerve changes r e s u l t from abnormalities of metabolism, u s u a l l y i n c h r o n i c a l l y unregulated diabetes.  L i s a et a l (1942) was unable to demonstrate pathological  changes i n the nerves of either diabetic or non-diabetic patients exhibited peripheral a r t e r i o s c l e r o s i s .  who  In diabetes a f a i l u r e to u t i l i z e  carbohydrates i n amounts adequate to s a t i s f y the patients needs i s followed by oxidation of protein and f a t to meet the necessary energy requirements, (Soskin and Levine, 1947). i v e f a t oxidation may  I t i s probable that the excess-  lead to peripheral demyelinization.  The phospho-  l i p i d , c h o l e s t e r o l , and cerebroside content of the nerves involved i n diabetic n e u r i t i s i s much lower than that of normal nerves, (Randall, 1938). Herstein and Weinrath (1945) i n agreement with other workers f i n d that an apparently s e l e c t i v e and frequently premature arierioscler-» osis occurs i n the p e r i p h e r a l vessels of the .lower extremeties i n  6 diabetic patients*  The increased incidence of a r t e r i o s c l e r o s i s i n these  patients, i r r e s p e c t i v e of the u s u a l l y accepted factors which determine a r t e r i o s c l e r o s i s i n non-diabetic persons, may  indicate a metabolic  o r i g i n or imply an acceleration of the process by some metabolic factoro From the s t a t i s t i c a l standpoint the r e l a t i o n s h i p between such factors and peripheral vascular disease i s not understood or a v a i l a b l e * The presence of cholesterol deposits i n the a r t e r i o s c l e r o t i c lesions and the hypercholesterolemia of patients with neglected or poorly controlled diabetes suggest a linkage between the two However, no such connection has so f a r been established*  disorders*  I t does not  follow that i n the treatment of diabetes such factors as c o n t r o l of weight and hypertension can be ignored or that suitable dietary and  insulin  regimens can be neglected. In reviewing 100 cases of diabetic neuropathy, Rudy and Epstein (1945) observed t h i s disorder not only i n acute stages of diabetes but also a f t e r the control of the glycosuria and hyperglycemia^ and i n chronic and even mild cases of diabetes« In summary, diabetic neuropathy may be considered a  generalized  neurologic disturbance which appears as part of the primary disease. appears most often i n poorly regulated patients.  It  Boyd et a l , (1942)  states that evidence presented supports the premise that -degenerative sequelae of diabetes mellitus represents l a t e compromise l e v e l s of diabetic control and that they can be anticipated i n the patient whose regimen of diabetic control or d i e t f a l l s appreciably short of physiologic l e v e l s , and that they can be prevented through the maintenance of suitable standards of management.  7  Alloxan Diabetes There are several ways to cause experimental diabetes mellitus i n laboratory animals.  " P i t u i t a r y diabetes" may be induced by the  i n j e c t i o n of anterior p i t u i t a r y extract (Selye, 1947)*  Large i n j e c t i o n s  of glucose w i l l cause hyperglycemia and eventually r e s u l t i n "glucose diabetes".  Both of these methods cause an increased functional demand  on the i s l e t s of Langerhans with the subsequent degeneration of the i s l e t t i s s u e . -Another method i s pancreatectomy which also removes the source of i n s u l i n .  However t h i s method i s almost impossible with small  animals" such as the r a t e s p e c i a l l y when the pancreas i s ' d i f f u s e .  By  f a r the most successful method to date i s an i n j e c t i o n of alloxan. In 1937 Jacobs (1937) was the f i r s t to show that the i n t r a venous i n j e c t i o n of alloxan i n t o rabbits caused a t r a n s i t o r y hyperglycemia followed by hypoglycemia, then diabetic convulsions and death* Shaw Dunn and h i s collaborators (1943) demonstrated that alloxan produces a selective necrosis of the i s l e t s i n rabbits*  Other investigators have  observed the same e f f e c t s on the i s l e t s and the development of persistent diabetes following parenteral i n j e c t i o n s of alloxan i n rabbits (Bailey and Bailey, 1943), white rats (Gomori and Goldner, 1943), hooded r a t s (Duff and S t a r r , 1944) and dogs (Goldner and Gomori, 1943). " In v i t r o , alloxan i n h i b i t s the conversion of glucose-l-phosphate to glucose-6-phosphate, the second step i n glycogen metabolism* believed t o destroy the phosphoglucomutase  enzyme system.  It i s  The i n t a c t  pyrimidine r i n g appears t o be e s s e n t i a l f o r i t s a c t i o n since several other related substances such as a l l o x a n t i n and d i a l u r i c a c i d have been found to be diabetogenic.  8 I t i s possible to detect alloxan i n the blood immediately i n j e c t i o n and within f i v e minutes i t i s a l l removede  after  However, i t s normal  presence i n the blood has not been d e f i n i t e l y establishedo  Glutathione,  a non-glucose reducing s u l f h y d r y l , i n t e r a c t s with alloxan and w i l l a c t u a l l y prevent diabetes i f injected before and during the alloxan injection. The exact mode of a c t i o n of alloxan i s s t i l l undetermined but i t has been demonstrated h i s t o l o g i c a l l y to cause i s l e t c e l l necrosis responsible f o r diabetes by the selective destruction of the Beta c e l l s . •Alloxan may be toxic to the Beta c e l l s or compete i n the enzyme systems present i n the c e l l s .  I t i s known, however, that i t does not act  through the p i t u i t a r y . Typical alloxan diabetes has been described by Duff and S t a r r (1944) i n hooded r a t s .  The c h a r a c t e r i s t i c fluctuations i n the blood  sugar l e v e l show the reaching of a peak, 150 to 240 mg %, within two hours a f t e r the i n j e c t i o n , followed by a f a l l below normal by 6 hours then a r i s e above normal within 6 to 24 hours.  The hypoglycemia d i d  not always occur and hypoglycemic convulsions were not observedo Glycosuria was present within f i r s t 24 hours i n a l l cases of marked hyperglycemia. A f t e r 24 hours the r e s u l t s varied with the dose.  With higher  dosages, 300 to 350 mg/kg, the blood sugar rose r a p i d l y to very high values, 800 to 1450 mg %  t  i n 30 t o 48 hours and death occurred i n a state  resembling diabetic coma i n 36 to 96 hours.  With lower dosages, 175 to  200 mg/kg» the blood sugar rose gradually to a peak of 500 mg % i n 2^ to 5 days and the animals survived f o r months with persistent diabetes. The blood sugar varied between 300 and 500 mg % and there was marked  9 glycosuria, p o l y u r i a , polydipsia, polyphagia and progressive loss of weight* Kennedy and Lukens (1944) made the observations that necrosis of the i s l e t s of the pancreas may be apparent i n 12 hours or l e s s *  They  also found that although the i n i t i a l hyperglycemia can be prevented by i n s u l i n or p h l o r h i z i n , t h i s does not protect the i s l e t tissue from necrosis, and that alloxan does not inactivate i n s u l i n i n v i t r o . Chronic hyperglycemia was produced i n 90 - 95% adult white rats starved f o r 48 t o 60 hours and then injected subcutaneously with 175 mg/kg alloxan as compared with 25% i n unstarved r a t s .  Feeding.glucose  6 hours previous to the i n j e c t i o n of alloxan diminished the susceptib i l i t y of the starved animals t o alloxan.  A l s o epinephrine administered  immediately p r i o r t o the alloxan protected the starved animals. However, B vitamins and ascorbic acid gave no protection when given 6 hours previous to alloxan i n j e c t i o n , (Kass and Waisbren, 1945)• In summary, the use of alloxan f o r the establishment of diabetes i n experimental animals i s one of the simplest and most e f f e c t i v e methods known and the type of diabetes may be considered t o be i d e n t i c a l with the diabetes mellitus found i n humans. The metabolism i n diabetic coma produced by alloxan has been studied by Kaplan, Franks and Friedgood (1945)•  Upon i n t r a p e r i t o n e a l  i n j e c t i o n of a single dose of 200 mg/kg i n t o f a s t i n g r a t s they produced a state of diabetic coma analogous to severe human coma.  In these  animals the plasma inorganic phosphate and blood sugar rose, l i v e r glycogen and t o t a l acid-soluble phosphate decreased.  An increase i n  l i v e r inorganic phosphate occurred with a concurrent f a l l i n adenosine* pyrophosphate and other organophosphate.  Very  large doses of i n s u l i n  1 0  caused improvement i n both the c l i n i c a l and chemical states of comatose rats. The f a c t that alloxan, a purine d e r i v a t i v e , can produce necrosis and atrophy of the beta c e l l s has l e d to the hypothesis that some s i m i l a r toxin may be responsible f o r human diabetes.  Recently, Conn and h i s  coworkers postulated that such a f a c t o r may be responsible f o r the  genesis  of pancreatic damage since they could demonstrate a -positive c o r r e l a t i o n between an increase i n endogenous purine metabolism and the development of temporary diabetes during the administration of adreno-corticotrophic hormone (ACTH) of the anterior p i t u i t a r y to human subjects.  It i s signifi-  cant that the temporary diabetes produced i n these subjects was r e s i s t a n t to the action of exogenous i n s u l i n , a phenomenon which cannot be attributed s o l e l y to a decrease i n the production of i n s u l i n by the pancreas,, since i t occurs also when ACTH i s administered  t o the alloxanized  rat. Opposed to t h i s view i s Houssay who  observes that i t i s  improbable that the anterior lobe of the p i t u i t a r y contains a substance whose function i t i s to destroy the i s l e t c e l l s .  The physiologic point of  view i s that the anterior lobe contains a hormone which counteracts  the  i n s u l i n i n some way and that i f t h i s hormone i s i n great excess, i t may cause such great a c t i v i t y i n the i s l e t c e l l s that they are overworked and die. Graham (1950) proposes the hypothesis that i n health there i s a good balance between the amount of i n s u l i n which i s necessary and active p r i n c i p l e of the anterior lobe.  the  I f the l a t t e r i s overactive,  more i n s u l i n w i l l have to be produced, and i f an i n s u f f i c i e n t amount i s produced, the blood sugar w i l l r i s e and a l l the symptoms of diabetes  will  11  appear*  The thyroid a l s o has an a f f e c t on i n s u l i n production, since when  the B«MoRo i s high i n toxic goiter, more i n s u l i n i s required and when i t i s low i n myxedema, l e s s i n s u l i n i s needed*  The adrenal cortex a l s o has  an influence on the amount of i n s u l i n needed, and i n Addison's disease spontaneous hypoglycemia may occur*  There i s another d i s t i n c t i o n which  i s u s e f u l i n c l i n i c a l p r a c t i c e , and that i s the one between the sensitive and i n s e n s i t i v e caseso  The former need very l i t t l e i n s u l i n and the  blood sugar decreases r a p i d l y * are  Fortunately the majority of diabetics  sensitive t o i n s u l i n but there are some who require large amounts*  One i n t e r e s t i n g f a c t about these cases i s that they are stable and are not l i k e l y ' t o have sudden attacks of hypoglycemias  Another group of  patients are d i f f i c u l t to treat e f f i c i e n t l y because t h e i r i n s u l i n requirements fluctuate widely e  I f the diabetes i s thought t o be due t o  the formation of very l i t t l e i n s u l i n , i t i s hard to imagine that the supply of i n s u l i n varies from day to day*  There i s some evidence that  the p i t u i t a r y gland can vary i n t h i s way*  I f the p i t u i t a r y can vary i t s  a c t i v i t y i n acromegaly, i t i s possible that i t can also vary the supply of hypothetical substance which i n t e r f e r e s with the i n s u l i n *  Materials and Methods Description of Experimental Animals The brains used i n t h i s i n v e s t i g a t i o n were from a group of r a t s used i n the study of diabetes mellitus and i t s relationship t o hypertension*  This work was carried out by Dr. 'A.L. Chute of the Sick Children's  Hospital, Toronto.  Diabetes was induced i n both male and female r a t s , age  1-1/2 t o 3 months, by a single intravenous i n j e c t i o n of alloxan with a dosage at 175 t o 200 mg/kg of body weight.  Both the diabetic animals and  the non-diabetic control animals were fed on an increased s a l t d i e t with sodium ohloride making up 10% of the d i e t * Only those rats which subsequently developed diabetes were used i n t h i s examination.,  Some of the rats were maintained on i n s u l i n while  others were unrestricted*  A f t e r the alloxan treatment the rats died at  varying i n t e r v a l s from two to f i v e months or were s a c r i f i c e d at the termination of the experiment* Fixation The r a t heads were severed from the body and immersed i n 10$ formalin f i x a t i v e *  The top of the s k u l l was removed t o permit complete  and quick f i x a t i o n of the brain* H i s t o l o g i c a l techniques Blocks of each b r a i n were embedded i n p a r a f f i n according to the  schedule i n L i l l i e (1948)*  Dehydration was carried out i n methyl  alcohol and the tissue cleared i n xylene*  Sections were cut on a Spencer  rotary microtome at 8 and 12 microns, the thicker sections being made f o r the  examination of large neurons*  The blocks of tissue and sections were  cut at varying i n t e r v a l s through the b r a i n to show cross-sections through the  cortex, cerebellum, medulla and v e n t r i c l e , including the choroid p l e x i * Among the h i s t o l o g i c a l techniques employed there was 1.  -  Harris Haematoxylin and eosin as a general oversight method* A s l i g h t v a r i a t i o n was to add 1$ potassium dichromate to the eosin.  This gave greater d i f f e r e n t i a t i o n of the red blood  c e l l s , staining them a b r i l l i a n t orange-red. 2*  Rapid one step Mallory-Heidenhain Trichrome s t a i n f o r connective tissue.  This procedure was found invaluable f o r demonstrating  12 .  collagen, h y a l l n , amyloid and various other connective tissue  . elements*  The staining s o l u t i o n - i s employed a f t e r hydration  of p a r a f f i n sections-and i s made up as follows: Phosphotungstic  1 gm  acid c r y s t a l s  Orange G (C. I* No 27)  2 gm  Analine blue, W.S. (C. I . No 707)  1 gm  Acid fuchsin (C. I . No 692)  3 gm 200 cc  D i s t i l l e d water 3*  Feulgen Stain*  Good r e s u l t s were obtained with this s t a i n only  when the t i s s u e had been i n the f i x a t i v e no longer than four months*  Any tissue which remained i n the 10^ formalin f o r  longer periods gave inconsistent staining*  In material f i x e d f o r  shorter periods the time of hydrolysis was found to be between 1-1/2 4*  t o 2 minuteso  Van Gieson s t a i n was employed i n conjunction with an alumhaematoxylin t o demonstrate h y a l i n i n f i l t r a t i o n *  5*  Weigert's Resorcin Fuchsin was used t o i l l u s t r a t e the e l a s t i c f i b r e s i n the walls of blood vessels* L i l l i e (1948), page 200,  This method, as found i n  gave excellent r e s u l t s *  I t was p a r t i c u -  l a r l y u s e f u l since any counterstain or s p e c i f i c s t a i n could be used complementary with 6.  ito  Masson's Trichrome s t a i n was also t r i e d but gave r e s u l t s i n f e r i o r to those obtained using the MaUory-Heidenhain Trichrome method with a much more elaborate procedure.  7*  Modified C a j a l technique employing bulk impregnation used f o r demonstrating  the nerve tissue elements*  method was Prolonged  f i x a t i o n did not appear t o e f f e c t the r e s u l t s of t h i s method.  13 Modified C a j a l Technique f o r s i l v e r staining blocks of t i s s u e * F i r s t the tissue was deformalinized  according to the method used by  Lhotka and F e r r e i r a , (1950): 1.  Wash blocks of formalin f i x e d tissue f o r 15 minutes i n d i s t i l l e d water*  2.  Transfer to 20% aqueous c h l o r a l hydrate solution f o r 24 hours. Repeat.  3.  Wash 15 minutes i n d i s t i l l e d water.  Modified C a j a l Technique 1*  Blocks of tissue overnight i n 100 ml. 95$ alcohol with 2-3  2*  drops of ammonium hydroxide*  D i s t i l l e d water 15 minutes.  Leave i n 2% aqueous s i l v e r n i t r a t e  f o r 16 - 24 hours* 3*  Solution renewed and continue staining f o r 14 t o 25 days.  4.  1 minute i n d i s t i l l e d water then reduce overnight i n 5% aqueous pyrogallol solution.  5.  Wash 15 minutes. Dehydrate i n graded alcohols, clear i n xylene, embed i n p a r a f f i n , section.  A t 12 microns, mount, clear i n xylene and cover using  balsam. Very good r e s u l t s were obtained by staining f o r 17 days i n the  silver  solution and reducing f o r about 10 hours* A. N i s s l Method f o r P a r a f f i n Sections using a Buffered Thionin  Staining  Solution Buffer Solution k»  Sodium Acetate  9.714 gm  Sodium Veronal  14.714 gm  D i s t i l l e d water t o make  500 ml  Buffer Solution B.  Sodium Chloride  42.5 gm  D i s t i l l e d water t o make  500 ml  Solution A. i s 1/7 molar with respect t o both sodium acetate and veronal,, To each 5 ml of solution k are added 2 ml of solution B^ a variable quantity (a ml) of n/10 HC1 and (18 - a) of carbon dioxide free d i s t i l l e d water. •• For pH of  a ml  18 ~ a ml  5.32  8  10  4.66  10  8  4.13  12  6  3.62  14  4  2.62  16  2  Use a pH meter to check the pH of the desired s o l u t i o n . Staining Solution  St00k solution  Thionin Water  Dilute staining solution  1 gm 100 ml  Buffer of chosen pH  10 ml  Stock solution  .5 ml  For n i s s l bodies of large motor neurons approximate pH o f 3*5 i s recommended, that i s with range between pH 4.5 and 3.  For cytoplasmic  processes of g l i a c e l l s e s p e c i a l l y from formalin f i x e d t i s s u e , approximate pH of 4.5 i s recommended, with a range between pH 5 and 4.  The s t a i n i n g  i s c a r r i e d out a t the water l e v e l f o r 10 minutes or longer at room temperature.  No overstaining w i l l occur.  The sections are rinsed i n  d i s t i l l e d water, dehydrated, cleared and mounted i n Canada Balsam. Among the other h i s t o l o g i c a l techniques used, but giving unsatisfactory r e s u l t s , was included!  The Periodic-Acid-^Schiff»s-Reagent (PAS) histopathologic technique of McManus (1948) t o indicate h y a l i n degeneration.  In pathologic states ,  c h a r a c t e r i s t i c coloring i s obtained of the hyalin of a r t e r i o s c l e r o s i s * The duration of f i x a t i o n was believed t o upset the hydrolysis by the periodic a c i d . Rio Hbrtega s s i l v e r carbonate method was attempted t o show possible l  astrocyte p r o l i f e r a t i o n but r e s u l t s were exceedingly poor since the tissue was not f i x e d i n formalin ammonium bromide.  Results and Observations  Gross Examination Upon gross examination of the i n t a c t r a t brains, the most s t r i k i n g change was observed i n the vascular system.  About one quarter  of the diabetic rats exhibited massive hemorrhages v i s i b l e on the external surface of the cerebral hemispheres and cerebellum.  Several  instances of subdural hemorrhage, presumably of venous o r i g i n , were noted. I t i s assumed that death was the r e s u l t of these vascular  disturbances,  e s p e c i a l l y i n the larger hemorrhages, since death occurred spontaneously i n these animals*  One of the c o n t r o l rats had a massive subarachnoid  hemorrhage between the cerebrum and cerebellum p o s s i b l y o r i g i n a t i n g from the Aqueduct of Sulvius. The external appearance of the r a t brains and the meninges were normal aside from the vascular  disturbances.  Upon d i s s e c t i o n of the brains, t o obtain blocks of t i s s u e suitable f o r h i s t o l o g i c a l embedding, numerous s u b c o r t i c a l lesions were observed (FIG. 1).  Blood c l o t s associated with the t h i r d and l a t e r a l  16 v e n t r i c l e s also were discovered and one instance of a large hemorrhage was found almost completely blocking the b r a i n stem i n the region of the pons« H i s t o l o g i c a l Examination The basic h i s t o l o g i c a l change consisted of an a l t e r n a t i o n i n the walls of the blood vessels culminating i n massive hemorrhage with r e s u l t i n g i n f a r c t i o n of the nervous tissue©  The vessels most severely  affected were the small p i a l a r t e r i e s and veins with t h e i r c o r t i c a l branches. The majority of hemorrhages were of venous o r i g i n (FIG. 2) or associated with the v e n t r i c l e s (FIG. 3 and 4) although i n the l a t t e r case the vessels of the choroid plexus u s u a l l y appeared normal.  Multiple m i l i a r y hemorr-  hages of a d i f f u s e nature were very common, occurring i n the cortex, basal n u c l e i and medulla (FIG. 5)»  I t i s possible that i n some instances the  large area of bleeding i s a r e s u l t of fusion of these c a p i l l a r y hemorrhages. As a consequence of the pathologic changes of the blood vessels, frequently, G i t t e r c e l l accumulations were noted i n the perivascular space (FIG. 6).  The invasions of these macrophagic c e l l s were more  frequent i n areas of s l i g h t diapedesis rather than massive hemorrhage. The reason f o r t h i s could be that massive bleeding probably  arose  spontaneously from the rupturing of a blood vessel and the animals died before neurologic changes could occur. A r t e r i a l Changes Using Weigert's e l a s t i c s t a i n technique i t was possible to show s p l i t t i n g and hypertrophy of the e l a s t i c f i b r e s i n the l a r g e r a r t e r i e s (FIG. 7)» purple  In the a r t e r i o l e s frequently the e n t i r e w a l l was  stained dark  i n d i c a t i n g that e l a s t i c hyperplasia was quite extensive.  The  e l a s t o s i s i s at f i r s t confined to the intima but eventually i t a l s o invades the media.  This i s i n d i c a t i v e of the conversion of a muscular  type of artery t o the e l a s t i c type designed t o withstand great s t r a i n such as present i n hypertension.  The amount of e l a s t i c tissue i n the  walls of an artery apprcacimately corresponds to the pressure of blood within i t . In the affected a r t e r i e s there was extensive p r o l i f e r a t i o n of the intima and the development of atheromatous areas.  In some cases an  accumulation or deposition of h y a l i n material was seen and i n the more severe examples, the lumen was considerably narrowed and sometimes comp l e t e l y o b l i t e r a t e d (FIG. 10).  Sharply defined n u c l e i were s t i l l d i s -  cernable i n regions of intense h y a l i n degeneration* Both the hyalin degeneration and the e l a s t i c hyperplasia described above are c h a r a c t e r i s t i c lesions of a r t e r i o s c l e r o s i s found i n the benign form of a r t e r i a l hypertension* Other c h a r a c t e r i s t i c lesions found were a r t e r i o l a r necrosis and c e l l u l a r hyperplasia. Necrosis was observed (FIG. 8) i n the smallera r t e r i e s involving either the whole of the v e s s e l or s p e c i f i c parts of i t . Occasionally red blood c e l l s were seen to i n f i l t r a t e the degenerated  areas  and diapedesis, accompanied by G i t t e r c e l l invasion, was also observed. Venous changes The majority of the massive,hemorrhages appeared t o occur from venous l e s i o n s .  Although i t was d i f f i c u l t t o distinguish necrosis of the  cerebral veins due t o the t h i n nature of the w a l l s , quite often h y a l i n degeneration was noted e s p e c i a l l y when bleeding was present (FIG. 9). The h y a l i n i n f i l t r a t i o n appeared not only i n the perivascular space but i n some instances advanced i n t o the surrounding nervous tissue of the b r a i n . In treated animals which did not exhibit excessive vascular dysfunction, a frequent observation was of congestion of the cerebral veins,  18 e s p e c i a l l y those i n the subarachnoid space*  I t was not possible to  ascertain whether the veins were distended beyond t h e i r normal diameter but i n some cases the walls appeared extremely t h i n f o r the size of the venous lumen* The veins of the s a l i v a r y glands which were also studied to a l e s s e r degree exhibited the above changes and i n one animal vacuolization was noted i n the media*  Fat s t a i n f a i l e d to determine whether there were  l i p i d deposits present* The venous congestion and necrosis, terminating i n cerebral hemorrhage again indicate the presence of a hypertensive condition* Though such findings were predominant i n the diabetic animals, there were examples found i n the control rats which were also fed the high s a l t d i e t . In summary, the a r t e r i o s c l e r o t i c and venous changes found i n t h i s study and s i g n i f y i n g hypertension i n the animals are i n complete agreement with the findings of Dr* A.L. Chute who noted such changes i n the kidneys, l i v e r and other organs of the same animals. Nervous System Changes As a r e s u l t of the pathologic changes of the blood vessels of the meninges and cerebral cortex there were circumscribed areas of destruction usually l i m i t e d to the cortex but occasionally involving the subjacent white matter.  Degeneration was also evident i n the medulla and  to a s l i g h t extent i n the cerebellum*  However, i n no case was there found  to be nerve tissue disturbance i n absence of vascular upset* One of the most s i g n i f i c a n t reactions to such vascular i n j u r y , fibrous thickening of the connective t i s s u e , was very common.  Selective'  staining demonstrated t h i s fibrous p r o l i f e r a t i o n to be h y a l i n i n nature and quite often,association with blood vessels was apparent*  However, i n  19 some animals these fibrous lesions were i n evidence i n the cbrtex (FIG. 11 and 12) where the only blood vessels were c a p i l l a r i e s . Using s i l v e r impregnation  methods, what appeared t o be areas of  softening were seen i n a l l parts of the brains of rats showing vascular disturbances.  This softening was quite d i s t i n c t since i t f a i l e d to s t a i n  and the only structures v i s i b l e were the c a p i l l a r i e s and scant connective tissue elements. In some of the alloxan d i a b e t i c animals, large neurons of the medullary n u c l e i were^degenerated.  Shrinkage was observed f a r i n excess  of that due t o the f i x a t i o n and embedding procedure.  The cytoplasm  became vacuolated considerably and the N i s s l granules  disappeared  (FIG. 14) and i n some c e l l s the n u c l e i were seen to be n e c r o t i c .  Sudan IV  s t a i n f a i l e d to show whether the vacuolization of the cytoplasm was due to l i p i d material. In the c o r t i c a l t i s s u e , changes were not too evident.  In one.  treated animal there were d i s t i n c t areas of the cortex which showed neuron degeneration  (FIG. 15). This was e s p e c i a l l y noticeable i n the polymorphous  c e l l l a y e r where the cytoplasm was necrotic and the n u c l e i swollen and vacuolated.  Associated with t h i s cytoplasmic break-down, neuronophagia  was seen by the great p r o l i f e r a t i o n of G i t t e r c e l l s about the c o r t i c a l cells. In summary, the nervous system complications i n the d i a b e t i c animals were always associated with concurrent disturbances.of the vascular system.  FIG. 1.  Spontaneous subarachnoid, hemorrhage on t h e l e f t w i t h m a s s i v e  cereTaral and s m a l l e r hemorrhages on the r i g h t e o s i n s t a i n , X 100  ).  ( H a e m a t o x y l i n and  FIG. 2 .  Spontaneous subarachnoid hemorrhage showing the necrosis o f  a small vein with resultant "bleeding into the subarachnoid space ( Haematoxylin and eosin stain, X 1 0 0 ) .  FIG. 3.  Hemorrhage into the l a t e r a l v e n t r i c l e ( haematoxylin and  eosin stein, X 120 ).  FIG. 4.  Hemorrhage from the l a t e r a l v e n t r i c l e into the cerebral t i s s u e .  The vessels o f the choroid plexus appear normal ( Safranin and f a s t green, X 100 ).  FIG. 5.  Multiple m i l i a r y hemorrhages o f the c a p i l l a r i e s i n the  medulla ( Trichrome stain, X 400 ).  FIG. 6.  Perivascular accumulation of G i t t e r c e l l s next to the  basal vein and smaller vessels which show some evidence o f diaped§sls ( Feulgen stain, X 800 ).  PIG. 7.  A meningeal artery showing s p l i t t i n g and hypertrophy of the  e l a s t i c f i b r e s and h y a l i n i z a t i o n of the media ( Weigert's r e s o r s i n fuchsin s t a i n , X 800).  FIG. 8.  Two examples of a r t e r i e s undergoing neorosis.  (a) Breakdown  of the externa, (b) Breakdown of e l a s t i c interna with opening i n wall, also G i t t e r o e l l invasion ( Trichrome s t a i n , X 100 ).  PIG. 9.  Middle oerebral vein with blood i n the perivascular spaoe  and h y a l i n i n f i l t r a t i o n into the surrounding t i s s u e ( van Gieson stain, X  200 ).  FIG. 10.  An oocluded meningeal artery e x h i b i t i n g extensive intimal  p r o l i f e r a t i o n and h y a l i n i n f i l t r a t i o n  ( Trichrome s t a i n , X 1000  ).  FIG. 11.  Seotion showing fibrous thickening of connective tissue i n  cerebral cortex and associated i n f a r o t i o n of the grey matter ( Masson's trichrome stain, X 200  ).  FIG. 12.  Selective s t a i n i n g i l l u s t r a t i n g h y a l i n nature of fibrous  i n f i l t r a t i o n i n cortex ( Van (Jieson stain, X 250  ).  FIG. 13.  Two  areas of softening i n the medulla showing only a loose  concentration of g l i a l struatures and c a p i l l a r i e s remaining Cajal s i l v e r stain, X 150  ).  ( Modified  F I G * 14.  Large degenerating motor neurons in the medulla showing  loss of F i s s l granules and necrosis of periphery of the oells ( ETissl stain, X 1000 ).  F I G * 15*  Polymorphous oells of the cortex exhibiting necrosis of the  cytoplasm with associated macrophages  ( Nisei stain, X 800 ).  20  Discussion  This reserach was carried on p r i m a r i l y as a h i s t o l o g i c a l i n v e s t i g a t i o n of the e f f e c t s of alloxan diabetes on the c e n t r a l nervous system.  In reviewing the recent l i t e r a t u r e on this subject the lack of  information i s evident.  In none of the nervous system manifestations of  diabetes mellitus i s there an agreement of opinion regarding the exact mechanism underlying the pathological changes.  In f a c t there i s s t i l l  much to be learned about the e t i o l o g y and pathology of neurological lesions i n diabetes. The s t r i k i n g feature of the r e s u l t s i n t h i s i n v e s t i g a t i o n i s the high incidence of vascular damage i n the experimental animals.  In the l a s t  ten years papers published concerning vascular dysfunction i n diabetes have been extensive and c o n t r o v e r s i a l .  Dolger (1950) gives a good review of  the subject, saying, "The suspected factors i n the development of premature vascular degeneration i n diabetes m e l l i t u s have included age, the control of glycosuria and hyperglycemia,  the use of i n s u l i n , the type of d i e t , and  the excess of c h o l e s t e r o l i n the d i e t with hypercholesterolemia."  The only  d e f i n i t e f a c t o r established as yet i s the duration of the disease. MLrsky (1946) claims i f the diabetic l i v e s long enough he w i l l develop one or another form of vascular disease.  In 1950 Warren claimed  never to have seen an autopsy of a diabetic whose disease l a s t e d f i v e years or more, free from a r t e r i o s c l e r o s i s , regardless of age.  Even i n  the young, P r i s c i l l a White (1941) admits the incidence of degenerative complications i n juvenile diabetes f a r exceeds expectation. substantiated by Chute (1948) i n h i s recent survey.  This i s  Arteriosclerosis  21  appears inevitablea Opposed to the above views are Hart and Lisa (1944) who admit arteriosclerosis i s very common though not consistent i n diabetes*  They  state the combined action of the diabetic and the age factors does not necessarily produce arteriosclerosis even i n cases of long duration. Of a more neutral stand i s Rabinowitch (1948), who has found arteriosclerotic disease of the heart, arteries and kidneys i s more common in the diabetic than in the non-diabetic, causing twice as many deaths as a l l other causes in diabetics*  It appears at an earlier age,  i s more severe and varies with duration, degree of control and severity of the diabetes. In experimental animals the picture i s somewhat different. The most satisfactory method for inducing experimental diabetes i s by administration  of alloxan.  However, this results in Beta -cell necrosis  of the islets of Langerhans i n the pancreas only, and there are none of the accompanying factors present.  Indeed, the primary factors  predisposing  an individual to diabetes mellitus are lacking. Bailey (1949) i n his review on alloxan diabetes states, "Iti s of considerable interest that neither diabetic neuritis nor arteriosclerosis has been observed in alloxan diabetic animals." Contrary to this, one year later, Beveridge and Johnson (1950,a) produced cardiovascular and renal lesions i n rats made diabetic with alloxan and maintained with insulin for periods ranging from 7 to 22 months. The lesions comprised myocarditis, marked chronic pyelonephritis and lesions involving the intima of coronary, renal and pancreatic arteries. They noted similar vascular and kidney lesion in control animals, but did not give the incidence of vascular upset i n either group.  22 . Added evidence to Beveridge and Johnson's-work i s obtained through the r e s u l t s of t h i s present i n v e s t i g a t i o n .  The vascular lesions  and cerebral hemorrhage c l e a r l y indicate the establishment of a r t e r i o s clerosis.  Although /the b r a i n i s one of the more common s i t e s f o r the  disease, Thoma ranks the cerebral a r t e r i e s fourth i n a l i s t , of f i f t e e n commonly elected locations, to the authors knowledge, a r t e r i o s c l e r o s i s has never been reported i n experimental diabetic animals before., There has been much speculation and l i t t l e conclusive evidence on the etiology of a r t e r i o s c l e r o s i s .  As long ago as 1926,  J o s l i n (1926)  thought that a factor i n the premature development of t h i s disease i n diabetes i s an excess of f a t i n the d i e t , i n the body tissues and i n the blood stream.  In diabetes the f a t and the cholestetol contents of the  blood are frequently elevated and this i s compatible with the p a t h o l o g i c a l physiology of a r t e r i o s c l e r o s i s .  I t should be remembered, however, that  diabetes i s p r i m a r i l y a disturbance of the metabolism of a number of substances and that the exact role of the increased sugar, f a t and chol e s t e r o l contents of the blood awaits a more complete study. In recent years the production of a r t e r i o s c l e r o s i s i n rabbits i s e a s i l y e l i c i t e d by administration of cholesterol*  Diabetic animals ..  exhibit a close relationship between blood sugar and blood l i p i d levels© P e t r u c c i o l i (1949) using alloxanized rabbits showed the l i p i d content of the blood increased i n the f i r s t hours following the i n j e c t i o n of alloxan, concomitant with the i n i t i a l hyperglycemia and showed a strong decrease i n the hypogrlycemic phase.  F i n a l l y a l l diabetic animals had an increased  blood l i p i d content. Continuing t h e i r experiments Beveridge and Johnson (1950,b) discovered the diabetic state had no e f f e c t on the l e v e l of plasma l i p i d s  23  in rats maintained with insulin, with the exception of an increase i n total cholesterol*  They also noted the presence of the diabetic state did  not effect any change i n the liver l i p i d pattern* • Unfortunately the plasma cholesterol values were unobtainable in this experiment, but i t i s presumed that they would be increased i n view of other results (Beveridge and Johnson)*  This i s interesting due to  the fact that Pollak (1945) obtained a generalized- atherosclerosis except for the cerebral vessels i n cholesterol fed rabbits*  When he ligated •  both carotid arteries, both jugular<veins or one artery and one vein, lesions of the cerebral vessels developed i n both the cholesterol-fed and the control animals* hypertensive*  These changes were not;atherosclerotic i n nature but  They were due to an impairment of the rate- of blood flow  and a change of the intravascular tension. Therefore, the diagnosis of arteriosclerosis and venous hemorrhage i n the brains of alloxanized rats, as found i n this experiment d e f i nitely indicates a hypertensive condition.  These findings are i n accord  with those of H. W. Radford who carried out a cytological investigation of the endocrine glands of the same experimental animals*. . The changes i n the cerebral veins are identical with those noted by Scheinker (1945), who, i n studying hypertension associated with massive cerebral hemorrhage, found two types of lesions.  The f i r s t was venous  congestion, resulting i n tremendous distention and the second was structural alterations of the vessel wall characterized by an extreme degree of atrophy and advanced signs of degeneration and necrosis* He draws attention to the predominantly venous origin of cerebral hemorrhage, which i s considered a terminal phase i n a sequence of events which begin with vascular disturbances. In the presence of advanced venular atrophy,.  24 elevations of venous pressure would appear to be an essential precursor to the massive escape of blood* Hicks and Warren cite venous stasis and necrosis, with or without capillary f r a g i l i t y as one of. the major causes of brain hemorrhage.* It is interesting to note that hypertensive diabetic patients show a 100$ increase i n capillary f r a g i l i t y over the normal and hypertensive nondiabetic patients and diabetic nonhypertensive patients showed a 53$ and 54$ increase respectively (Beaser et a l . , 1944)*  Evidently both diabetes  and hypertension are factors predisposing to vascular dysfunctions * . The hypertension observed i n these animals i s important due to the fact that such a condition has not been noted i n diabetic experimental animals to so great a degree before*  Since the diabetes i s not the  inducing factor the only other possibility i s the increased salt diet which was fed to both the alloxanized and control animals*  This would  account for the vascular changes observed i n the control rats*  It appears  therefore that salt-feeding alone can promote hypertension and this condition i s enhanced or aggravated by the presence of diabetes* The findings of Perera and Blood (1946) on patients with uncomplicated hypertensive vascular disease and various sodium chloride diets indicate that high blood pressure i s a disturbance i n the salt, and water metabolism referable to renal changes probably mediated by the adrenal cortex*  Schroeder (1948) also considers the possible role of the adrenal  cortex i n arterial hypertension and i t s relation to disturbances i n salt balance, however, conclusive results are s t i l l obscure.  . „  Possibly the increased salt i n the blood promotes an increase in the blood volume with a compensating increase i n pressure by the hearto The sodium and chloride ions may also affect the membrane permeability of  25 the various tissues involved since some investigators believe that riot only diabetes m e l l i t u s but a l s o hypertension i s a r e s u l t i n the membrane permeability of the c e l l s *  I t would be i n t e r e s t i n g to see the r e s u l t s of  further work where a close watch was kept on the plasma sodium and chloride content of s a l t - f e d animals t o determine the rate of excretion*  Quite  l i k e l y changes as a r e s u l t of increased d i e t a r y s a l t f o r short periods of time are r e v e r s i b l y u n t i l the c h a r a c t e r i s t i c vascular lesions have occurred. Nervous system manifestations i n the alloxanized diabetic animals occurred as a r e s u l t of the disturbances i n the blood supply to the b r a i n . J  This follows from the work of DeJong (1950) who states that i t i s becoming recognized with increasing frequency that many of the pathological conditions of the nervous system may be related d i r e c t l y t o the - vascular changes that are almost i n v a r i a b l y present i n diabetes*  The present-day treatment  of diabetes has f a i l e d to avert the vascular damage associated with diabetes and hence the complications i n the nervous system are becoming more prevalent. Probably a l l the damage t o the nerve c e l l s i s due to the anoxia r e s u l t i n g from the disrupted blood supply*  The neuron degeneration i n  the medulla appears to be the a f t e r - e f f e c t of peripheral neuropathy, since demyelination  occurs i n the extremities and such nerves are connected to  motor c e l l s i n the medulla.  26  Summary  Histological sections of the brains of alloxanized diabetic and non-diabetic rats fed on an increased salt diet were studied* The outstanding changes were evident in the vascular system of the brainso Hyalin degeneration and elastic hyperplasia indicated the presence of arteriosclerosiso Venous congestion and necrosis with resulting cerebral, hemorrhage, including the arterial dysfunction, indicated a hypertensive condition present in the animals* Secondary to these vascular changes and apparently a result of them, degeneration was noted in medullary neurons and to a lesser extent in the cortex. There appeared to be no difference in the pathologic effects on the vascular or nervous systems in diabetic rats maintained on insulin or those rats whose diabetes was uncontrolled* These observations were not confined to experimental animals alone since the non-diabetic.rats exhibited vascular disturbances but. to a much lesser extent* These results are in accord with those of Dr. A*L. Chute and.H.W* Radford who made similar studies on the same experimental animals of the endocrine glands and various other organs*  27  Conclusions  The m e l l i t u s  are  v a s c u l a r  d i a b e t i c  secondary  appears  animals,  diabetes,  than  diabetes  From d e f i n i t e l y  system  the  complications  p a t h o l o g i c a l  that  a r t e r i o s c l e r o s i s maintained  non-diabetic  i s  b e l i e v e d b e i n g  t h i s  t o  one  of  the  on  ratse  hasten the  experiment,  increases  state  of  diabetes  occurring  i n  the  an  incidence  the  may  common  or  w i t h  be  due  processes prevalent  increased of  more  i n s u l i n This  most  i s  of  i n  u n c o n t r o l l e d t o  the  f a c t  a g i n g ,  symptoms.  sodium  hypertension  c h l o r i d e  d i e t  e s p e c i a l l y  i n  animals. The  animals  t o  e i t h e r  i n  a r t e r i o s c l e r o s i s  d i a b e t i c  nervous  system. I t  that  c e n t r a l  might  high be  incidence  due  t o  an  of  hemorrhage  increase  i n  i n  hypertensive  c a p i l l a r y  f r a g i l i t y .  d i a b e t i c  28  Bibliography  Bailey, C C , 1949, Vitamins and Hormones, V o l . VII, New York Academic Press Inc., 365 p . Bailey, C C . and Bailey, O.T., 1943, The Production of Diabetes Mellitus i n Rabbits with Alloxan, J . Am. Med. Assn., 122 :  TC53:  Beaser, S.B., Rudy, A. and Seligman, A.M., 1944, C a p i l l a r y F r a g i l i t y i n Relation to Diabetes M e l l i t u s , Hypertension and Age, Arch. Int. Med., 73 t 18 - 22. Beveridge, J.M.R. and Johnson, S.E., 1950, a. Studies i n Diabetic Rats, B r i t . J . E x p t l . path., 31 : 285* Beveridge, J.M.R. and Johnson, S.E.., 1950, b . Studies i n Diabetic Rats, B r i t . J . E x p t l . path., 31 : 294* Boyd, J.D., Jackson, R.L. and A l l e n , J.H., 1942, Avoidance of Degenerative Lesions i n Diabetes Mellitus, J.A.M.A.,  118 : 694 - 696.  Chute, A.L., 1948, Survey of Patients with Juvenile Diabetes M e l l i t u s , Am. J . D i s . Child., 75 : 1 - 10. Dejong, R., 1950, Nervous System Complications i n Diabetes Mellitus, J . Nervous & Mental D i s . , I l l : 91 - l80T Dolger, H., 1947, C l i n i c a l Evaluation of Vascular Damage i n Diabetes Mellitus, J.A.M.A., 134 : 1289 - 1291. Dolger, H., 1950, Factors Influencing Premature Cardio-vascular Degeneration i n Diabetes Mellitus, Progress i n C l i n i c a l Endocrinology, Soskin, S. e d i t o r , New York, Grune and Stratton. Dry, T.J. and Hines, E.A., 1941, The Role of Diabetes i n the Development of Degenerative Vascular Disease, Ann. I n t . Med., 14 : 1893. Duff, G.L. and Starr, H., 1944> Experimental Alloxan Diabetes i n Hooded Rats, Proc. Soc. Exper. B i o l . & Med., 57 : 280. Dunn, J.S., Sheehan, HoL. and McLetchie, N.G.B., 1943, Necrosis of I s l e t s of Langerhans Produced Experimentally, Lancet,  1 : 484.  29 Goldner, M.G. and Gomori, G., 1943, Alloxan Diabetes i n the Dog, Endocrinology, 33 .* 297* Gomori, G. and Goldner, M.G., 1943, Proc. Soc. Exp. B i o l . & Medicine, 54 : 287. Graham, G., 1950, Hypothesis f o r the Cause of Diabetes M e l l i t u s , The Harben Lectures of 1949, No. 2, J . Roy. Inst. Pub. Health and Hyg., 13 : 263 - 280. Harris^ W., 1922, Multiple Peripheral N e u r i t i s , Lancet, 2 : 8 4 9 . Hart, J.F. and L i s a , J.R., 1944, Diabetes Mellitus and A r t e r i o s c l e r o s i s , E f f e c t of Duration and Severity on A r t e r i a l Changes, New York State J . Med., 44 : 2479'- 2482. Herzstein,. .J• and l e i n r o t h , L»A •, 1945," A r t e r i o s c l e r o t i c Peripheral Vascular Disease i n Diabetes, Arch. Int. Med., 7 6 : 34 - 38. Hicks, S.P. and Warren, S., 1950> Introduction t o Neuropathology, New York, McGraw-Hill Book Co., 1st ed. ~ J o s l i n , E.P., 1926, A r t e r i o s c l e r o s i s and Diabetes, Ann. C l i n . Med., 5 : 1061. Jordan, W.R., 1936, Neuritic Manifestations i n Diabetes M e l l i t u s , Arch. Int. Med.j 57 : 307. Jacobs, H.R., - 1937, Hypoglycemic A c t i o n of Alloxan, Proc. Soc. Exp. Biol., and Med., 37 :-407. Kaplan, N.O., Franks, M. and Friedgood, C.E., 1945, Metabolism i n Diabetic Coma Produced by Alloxan, Science, 102 : 4 4 7 . Kass, E.H.'and Waisbren, B.A., 1945, A Method f o r Consistent Induction of Chronic Hyperglycemia with Alloxan, Proc. Soc. Exp.. B i o l , and Med., 60 : 303 - 306. Kennedy, W.B. and Lukens, F.D.W.,' 1944> Observations on Alloxan Diabetes, Proc. Soc. Exp. B i o l , and Med., 57 : 143. L i l l i e , R.D., 1948, Histopathologic Technic, 1st ed., Philadelphia, The Blakiston Company. :  L i s a , J.R., Magiday, "M. and Hart, J.F., 1942, Peripheral A r t e r i o . s c l e r o s i s i n Diabetic and Non-diabetic, J.A.M.A., 118 : 1353 - 1356, " !  McManus, J.F.A., 1948, Histoohemical Uses of Periodic Acid, Amer. J . * Pathology, 24 ": 643. :  30  Mirskey, I.A., 1946, Our,Challenge f o r the Future, Diabetes Abst., 5 : 71. Needles, ¥., 1930, Vitamin Studied i n Cases of Diabetic N e u r i t i s , Arch. Neurol, and Psychiat., 41 : 1222. Perera, C-.A. and Blood, D.W., 1946, Hypertension, A Disturbance i n S a l t and Water Metabolism, Am. J . Med.,-1 s 602 - 606 o  P e t r u c c i o l i , L., 1949, The Behaviour of the L i p i d Content of the Blood .in the Experimental Alloxan Diabetes, Arch..E» '. ~ Maragliano pat. e C l i n . , 4 : 705 - 726. Pollak, O.J., 1945, Attempts t o Produce Cerebral Atherosclerosis, Arch. Pathology, 39 s 16 - 21. ~ ~ • Rabinowitch,-I.M., 1948, Relationship betvreen Impairment of L i v e r Function and Premature Development of A r t e r i o s c l e r o s i s i n Diabetes M e l l i t u s , Canad. M. A. <J», 58 : 547 - 556. Randall, L.O., 1938, Changes i n L i p i d Composition of Nerves from A r t e r i o s c l e r o t i c and Diabetic patients, J . B i o l . Chem., 125 : 723. ~~~ ' , Rudy, A« and Epstein, S.H., 1945, Review of l o o Cases of Diabetic Neuropathy Followed, from One to 10 years, J . C l i n . Endocrinol., 5 : 92 - 98. Rundles, R.W.,: 1950, Diabetic Neuropathy, B u l l . N.Y. Acad. Med., 26 :^598 - 616. Rundles, R.W., 1945,.Editorial, J.A.M.A., 128 : 1230. Scheinker, I.M., 1945, Changes i n cerebral Veins i n Hypertensive Brain Disease and t h e i r Relation to Cerebral Hemorrhage, C l i n i c a l Pathologic Study. Arch. Neurol, and Psychiat., 54 s 395.,. . Schroeder,^H.A., 1948, Low Salt Diets and A r t e r i a l Hypertension, Am. J . Med., 4.: 578. ' ' ' . Selye, H., 1947, Textbook of Endocrinology, Montreal, Acta Endocrinological Soskin, S. and Levine, R., 1947, .Carbohydrate Metabolism, 1st ed..., Chicago, The University of Chicago Press., \ :  Treusch, J.V., 1945, Diabetic Neuritis, Proc. Staff Meet., Mayo C l i n . , 20 : 393. Warren, S., 1938, The Pathology of Diabetes M e l l i t u s , 2nd ed., Philadelphia, Lea and Febriger.  31  YJhite, P.,; 1941, Diabetes i n Youth, New Eng. Jo Medo, 224 : 586.  

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