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

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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 this thesis as conforming to the standard required from candidates for the Degree of MASTER OF ARTS l£3 3? Members of the Department of Biology The University of British Columbia A p r i l , 1951 Abstract A, group of alloxanized rats fed on a high salt diet were studied histologically to determine the direct or indirect effect of diabetes mellitus on the nervous systems The most significant changes observed were i n the vascular system of the brain* Lesions of the arteries were identical with those found i n arteriosclerosis and massive hemorrhages accompanied by multiple diffuse diapedesis not only i n the arteries but also i n the veins was indicative of hypertension* It i s believed the hypertensive condition was induced by a combination of the diabetes and salt feeding. Associated 'with the vascular dysfunction generalized cerebral changes were noted. They included neuron degeneration, exhibited by loss of Nissl granules^ vacuolization of the cytoplasm and cerebral softening. Also Gitter c e l l invasion and proliferation occurred at the sites of vascular disturbance. It appears that the neurological involvement i n diabetes i s a direct result of changes in the blood system and not of the disease i t s e l f . i i Acknowledgements The author wishes to express his thanks to Dr. A.H. Hutchinson, under whose direction this research was carried out. A. particular debt of gratitude i s owing for the invaluable fundamental histological methods and procedures I have learned i n our association as professor and student. I am indebted also, to Dr. W.C. Gibson who suggested the problem and was responsible for obtaining the experimental material. Without Dr. Gibson's patient supervision, the silver techniques for nervous tissue would never have succeeded. To Dr. J.A» Allardyce, for his kind advice and helpful suggestions, I am most grateful. His teachings i n physiology have done much to elucidate the problem i n this aspect* thanks go to Dr. A»L. Chute, who supplied the experimental material as a supplementary problem i n conjunction with his own work on diabetes and hypertension. i i i Table of Contents Page Abstract . . . . . . . . . . i Acknowledgements • i i Table of Contents • . i i i Introduction . . . . . . . . . . . . . . 1 Historical Review . • 2 Diabetic Neuropathy . . . . . . . . . . . . 3 Alloxan Diabetes . • . 7 Materials and Methods . • • • » 11 Results and Observations 15 Gross. Examination 15 Histological Examination . . . . . 16 Discussion * . « . « • • • » . . . . . . . . . . . 20 Summary • . . . . . . . . . . . . . . . . » • • • 26 Conclusions . . . . . . . . . . . . . . . . . . . 27 Bibliography . . . . . . . 28 Introduction The nervous tissue of alloxanized rats was studied to determine the direct or indirect effect of diabetes on the central nervous system. The diabetic syndrome i s primarily a disturbance i n the regulation of 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^ The central nervous system complications of diabetes include involvement of the peripheral nerves, spinal cord, brain and autonomic nervous system, as well as those pathologic changes •which develop i n association with the hypoglycemic state• Arteriosclerosis i s an extraordinarily frequent accompaniment of diabetes and i t may well be that many of the above-^nentioned complications, as well as syndromes of both focal and diffuse cerebral involvement may develop secondary to disease of the cerebral and meningeal arteries« It i s the purpose of this problem to f i r s t investigate the vascular system of the brain using general and specific staining procedures and then to examine the nervous tissue for pathologic changes by means of specific silver staining methodso A-preliminary research by Dr. AoL» Chute has indicated the presence of arteriosclerosis as well as a hypertensive condition i n other parts of the experimental animals including the kidney, l i v e r and pancreas* 1 Historical Review The cause of diabetes mellitus i s unknown. The relationship between diabetes and arteriosclerosis, which frequently are found together, i s obscure. The effect of diabetes on the nervous system i s as yet unsatisfactorily explained. It i s well known that nervous system complications may occur in association with diabetes mellitus. Certain of these coexisting neurologic manifestations appear frequently, and they are easily recognized from a c l i n i c a l point of view. Others are less often encountered and consequently many observers may not interpret them as nervous system complications of this disease. Often i t i s stated that the diabetic process has a special predilection for 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 limited to the peripheral portions of the nervous system, and involvement of the spinal cord, brain stem, midbrain and cerebrum, as well as the autonomic nervous system, has been observed. In fact, there may be a diffuse affection of the entire nervous system, including both the central and peripheral portions. The part played by vascular changes, especially 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 brain 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 significant. White described dilation 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 proliferation, and displacement of the ependyma. It must be recalled, however, that these postmortem changes must be differentiated from those alterations of structure which may be the result of vascular damage, hypoglycemia and infection. 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 light of current observation* It i s to be recognized rather as an integral manifestation of the basic disorder, an associated concomitant of the disease. Such a biological approach to this problem was postu-lated by Dry and Hines (1941) on the basis of "an inherent weakness which affects both the insulin-producing tissues and the vascular system" with a common mechanism for the production of arteriosclerosis, retino-pathy and neuropathy on the basis of involvement of the nutrient vessels of these structures. The diabetic animal provides ideal material for 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 neuritis, i s the neurological complication most frequently associated with diabetes mellitus. It was f i r s t described i n 1864 by Calvi who observed pains i n the distribution of the sciatic nerves along with areas of anesthesia i n diabetes. 4 He suggested that the neurologic lesions were the result rather than the cause of the disorder* Since.thenj there have been many c l i n i c a l des-criptions of the peripheral nerve changes that may occur i n diabetes• The manifestations of diabetic neuropathy are identical with other types of peripheral neuritis* There i s pain, paresthesias, flaccid pareses and ataxia. Also disturbances of cutaneous and proprioceptive sensations, reflex changes and loss of motor power. There have been several attempts to classify the peripheral neuropathy associated with diabetes mellitus. Jordan (1936) has suggested the following categories: 1. The hyperglycemic type i s characterized mainly by tenderness of the nerves. 2. The circulatory type i s manifested by symptoms such as pain and paresthesis and signs such as hyporeflexia and hyperesthesia. 3« The degenerative type begins insidiously and progresses slowly over a period of years. 4. The neuritic type, which i s the commonly described diabetic neuritis, i s marked by pain and paresthesias and there i s weakness which i n some cases progresses to paralysis* Treusch (1945) has proposed a similar classification -with an additional division, "diabetic visceral neuritis 1 1} which includes the cases of autonomic nervous system involvement. Patients have a burning paresthesias of the feet and usually 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. It i s more common i n older patients but not uncommon i n the young. The pathological changes 5 are generally a demyelinization of peripheral nerves« Harris (1922) has indicated the close similarity with beriberi and so-called alcoholic neuritis and suggested a vitamin B deficiency could be responsible. However, i t has been found that the neuropathy may develop i n patients who have been on an adequate diet. (Needles, 1930). Atherosclerosis has been cited by various workers as of pos-sible significance i n the development of dysfunction of the peripheral nerves, due to the resultant ischemia and anoxia of the nerves. However, once again, diabetic neuropathy may occur i n young persons who show no apparent peripheral arteriosclerosis. It i s probable that neither tMamine deficiency nor vascular disease i s alone responsible for the nervous iiupset and i t may be that the peripheral nerve changes result from abnormalities of metabolism, usually i n chronically unregulated diabetes. Lisa et a l (1942) was unable to demonstrate pathological changes i n the nerves of either diabetic or non-diabetic patients who exhibited peripheral arteriosclerosis. In diabetes a failure to u t i l i z e carbohydrates i n amounts adequate to satisfy the patients needs i s followed by oxidation of protein and fat to meet the necessary energy requirements, (Soskin and Levine, 1947). It i s probable that the excess-ive fat oxidation may lead to peripheral demyelinization. The phospho-l i p i d , cholesterol, and cerebroside content of the nerves involved i n diabetic neuritis i s much lower than that of normal nerves, (Randall, 1938). Herstein and Weinrath (1945) i n agreement with other workers find that an apparently selective and frequently premature arierioscler-» osis occurs i n the peripheral vessels of the .lower extremeties i n 6 diabetic patients* The increased incidence of arteriosclerosis i n these patients, irrespective of the usually accepted factors which determine arteriosclerosis i n non-diabetic persons, may indicate a metabolic origin 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 relationship between such factors and peripheral vascular disease i s not understood or available* The presence of cholesterol deposits i n the arteriosclerotic lesions and the hypercholesterolemia of patients with neglected or poorly controlled diabetes suggest a linkage between the two disorders* However, no such connection has so far been established* It does not follow that i n the treatment of diabetes such factors as control 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 this disorder not only i n acute stages of diabetes but also after 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. It appears most often i n poorly regulated patients. Boyd et a l , (1942) states that evidence presented supports the premise that -degenerative sequelae of diabetes mellitus represents late compromise levels of diabetic control and that they can be anticipated i n the patient whose regimen of diabetic control or diet f a l l s appreciably short of physiologic levels, 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. "Pituitary diabetes" may be induced by the injection of anterior pituitary extract (Selye, 1947)* Large injections of glucose w i l l cause hyperglycemia and eventually result 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 tissue. -Another method i s pancreatectomy which also removes the source of insulin. However this method i s almost impossible with small animals" such as the rat especially when the pancreas is'diffuse. By far the most successful method to date i s an injection of alloxan. In 1937 Jacobs (1937) was the f i r s t to show that the i n t r a -venous injection of alloxan into rabbits caused a transitory hyper-glycemia followed by hypoglycemia, then diabetic convulsions and death* Shaw Dunn and his 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 effects on the i s l e t s and the development of persistent diabetes following parenteral injections of alloxan i n rabbits (Bailey and Bailey, 1943), white rats (Gomori and Goldner, 1943), hooded rats (Duff and Starr, 1944) and dogs (Goldner and Gomori, 1943). " In vitro , alloxan inhibits the conversion of glucose-l-phosphate to glucose-6-phosphate, the second step i n glycogen metabolism* It i s believed to destroy the phosphoglucomutase enzyme system. The intact pyrimidine ring appears to be essential for i t s action since several other related substances such as alloxantin and dialuric acid have been found to be diabetogenic. 8 It i s possible to detect alloxan i n the blood immediately after injection and within five minutes i t i s a l l removede However, i t s normal presence i n the blood has not been definitely establishedo Glutathione, a non-glucose reducing sulfhydryl, interacts with alloxan and w i l l actually prevent diabetes i f injected before and during the alloxan injection. The exact mode of action of alloxan i s s t i l l undetermined but i t has been demonstrated histologically to cause i s l e t c e l l necrosis responsible for 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 . It i s known, however, that i t does not act through the pituitary. Typical alloxan diabetes has been described by Duff and Starr (1944) i n hooded rats. The characteristic fluctuations i n the blood sugar level show the reaching of a peak, 150 to 240 mg %, within two hours after the injection, followed by a f a l l below normal by 6 hours then a rise above normal within 6 to 24 hours. The hypoglycemia did 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. After 24 hours the results varied with the dose. With higher dosages, 300 to 350 mg/kg, the blood sugar rose rapidly to very high values, 800 to 1450 mg % t i n 30 to 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 for months with persistent diabetes. The blood sugar varied between 300 and 500 mg % and there was marked 9 glycosuria, polyuria, 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 less* They also found that although the i n i t i a l hyperglycemia can be prevented by insulin or phlorhizin, this does not protect the i s l e t tissue from necrosis, and that alloxan does not inactivate insulin i n v i t r o . Chronic hyperglycemia was produced i n 90 - 95% adult white rats starved for 48 to 60 hours and then injected subcutaneously with 175 mg/kg alloxan as compared with 25% i n unstarved rats. Feeding.glucose 6 hours previous to the injection of alloxan diminished the suscepti-b i l i t y of the starved animals to alloxan. Also epinephrine administered immediately prior to the alloxan protected the starved animals. However, B vitamins and ascorbic acid gave no protection when given 6 hours previous to alloxan injection, (Kass and Waisbren, 1945)• In summary, the use of alloxan for the establishment of diabetes i n experimental animals i s one of the simplest and most effective methods known and the type of diabetes may be considered to be identical 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 intraperitoneal injection of a single dose of 200 mg/kg into fasting rats 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 total acid-soluble phosphate decreased. An increase i n li v e r inorganic phosphate occurred with a concurrent f a l l i n adenosine* pyrophosphate and other organophosphate. Very large doses of insulin 1 0 caused improvement i n both the c l i n i c a l and chemical states of comatose rats. The fact that alloxan, a purine derivative, can produce necrosis and atrophy of the beta cells has led to the hypothesis that some similar toxin may be responsible for human diabetes. Recently, Conn and his coworkers postulated that such a factor may be responsible for the genesis of pancreatic damage since they could demonstrate a -positive correlation 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 pituitary to human subjects. It i s s i g n i f i -cant that the temporary diabetes produced i n these subjects was resistant to the action of exogenous insulin, a phenomenon which cannot be attributed solely to a decrease i n the production of insulin by the pancreas,, since i t occurs also when ACTH i s administered to the alloxanized rat. Opposed to this view i s Houssay who observes that i t i s improbable that the anterior lobe of the pituitary 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 insulin i n some way and that i f this hormone i s i n great excess, i t may cause such great ac 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 insulin which i s necessary and the active principle of the anterior lobe. If the latter i s overactive, more insulin w i l l have to be produced, and i f an insufficient amount i s produced, the blood sugar w i l l rise and a l l the symptoms of diabetes w i l l 11 appear* The thyroid also has an affect on insulin production, since when the B«MoRo i s high i n toxic goiter, more insulin i s required and when i t i s low i n myxedema, less insulin i s needed* The adrenal cortex also has an influence on the amount of insulin needed, and i n Addison's disease spontaneous hypoglycemia may occur* There i s another distinction which i s useful i n c l i n i c a l practice, and that i s the one between the sensitive and insensitive caseso The former need very l i t t l e insulin and the blood sugar decreases rapidly* Fortunately the majority of diabetics are sensitive to insulin but there are some who require large amounts* One interesting fact about these cases i s that they are stable and are not likely'to 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 their insulin requirements fluctuate widely e If the diabetes i s thought to be due to the formation of very l i t t l e insulin, i t i s hard to imagine that the supply of insulin varies from day to day* There i s some evidence that the pituitary gland can vary i n this way* If the pituitary can vary i t s activity i n acromegaly, i t i s possible that i t can also vary the supply of hypothetical substance which interferes with the insulin* Materials and Methods Description of Experimental Animals The brains used i n this investigation were from a group of rats used i n the study of diabetes mellitus and i t s relationship to hyper-tension* 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 rats, age 1-1/2 to 3 months, by a single intravenous injection of alloxan with a dosage at 175 to 200 mg/kg of body weight. Both the diabetic animals and the non-diabetic control animals were fed on an increased salt diet with sodium ohloride making up 10% of the diet* Only those rats which subsequently developed diabetes were used i n this examination., Some of the rats were maintained on insulin while others were unrestricted* After the alloxan treatment the rats died at varying intervals from two to five months or were sacrificed at the termination of the experiment* Fixation The rat heads were severed from the body and immersed i n 10$ formalin fixative* The top of the skull was removed to permit complete and quick fixation of the brain* Histological techniques Blocks of each brain were embedded i n paraffin 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 for the examination of large neurons* The blocks of tissue and sections were cut at varying intervals through the brain to show cross-sections through the cortex, cerebellum, medulla and ventricle, including the choroid plexi* Among the histological techniques employed there was -1. Harris Haematoxylin and eosin as a general oversight method* A slight variation was to add 1$ potassium dichromate to the eosin. This gave greater differentiation 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 stain for connective tissue. This procedure was found invaluable for demonstrating 12 . collagen, hyalln, amyloid and various other connective tissue . elements* The staining solution-is employed after hydration of paraffin sections-and i s made up as follows: Phosphotungstic acid crystals 1 gm 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 Di s t i l l e d water 200 cc 3* Feulgen Stain* Good results were obtained with this stain only when the tissue had been i n the fixative no longer than four months* Any tissue which remained i n the 10^  formalin for longer periods gave inconsistent staining* In material fixed for shorter periods the time of hydrolysis was found to be between 1-1/2 to 2 minuteso 4* Van Gieson stain was employed i n conjunction with an alum-haematoxylin to demonstrate hyalin i n f i l t r a t i o n * 5* Weigert's Resorcin Fuchsin was used to il l u s t r a t e the elastic fibres i n the walls of blood vessels* This method, as found i n L i l l i e (1948), page 200, gave excellent results* It was particu-l a r l y useful since any counterstain or specific stain could be used complementary with ito 6. Masson's Trichrome stain was also tried but gave results inferior to those obtained using the MaUory-Heidenhain Trichrome method with a much more elaborate procedure. 7* Modified Cajal technique employing bulk impregnation method was used for demonstrating the nerve tissue elements* Prolonged fixation did not appear to effect the results of this method. 13 Modified Cajal Technique for silver staining blocks of tissue* F i r s t the tissue was deformalinized according to the method used by Lhotka and Ferreira, (1950): 1. Wash blocks of formalin fixed tissue for 15 minutes i n d i s t i l l e d water* 2. Transfer to 20% aqueous chloral hydrate solution for 24 hours. Repeat. 3. Wash 15 minutes i n d i s t i l l e d water. Modified Cajal Technique 1* Blocks of tissue overnight i n 100 ml. 95$ alcohol with 2 - 3 drops of ammonium hydroxide* 2* D i s t i l l e d water 15 minutes. Leave i n 2% aqueous silver nitrate for 16 - 24 hours* 3* Solution renewed and continue staining for 14 to 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 paraffin, section. At 12 microns, mount, clear i n xylene and cover using balsam. Very good results were obtained by staining for 17 days i n the silver solution and reducing for about 10 hours* A. Nissl Method for Paraffin Sections using a Buffered Thionin Staining Solution Buffer Solution k» Sodium Acetate 9.714 gm Sodium Veronal 14.714 gm Di s t i l l e d water to make 500 ml Buffer Solution B. Sodium Chloride 42.5 gm Di s t i l l e d water to make 500 ml Solution A. i s 1/7 molar with respect to 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 solution. Staining Solution St00k solution Thionin 1 gm Water 100 ml Dilute staining solution Buffer of chosen pH 10 ml Stock solution .5 ml For n i s s l bodies of large motor neurons approximate pH of 3*5 i s recom-mended, that i s with range between pH 4.5 and 3. For cytoplasmic processes of glia cells especially from formalin fixed tissue, approximate pH of 4.5 i s recommended, with a range between pH 5 and 4. The staining i s carried out at the water level for 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 histological techniques used, but giving unsatisfactory results, was included! The Periodic-Acid-^Schiff»s-Reagent (PAS) histopathologic technique of McManus (1948) to indicate hyalin degeneration. In pathologic states , characteristic coloring i s obtained of the hyalin of arteriosclerosis* The duration of fixation was believed to upset the hydrolysis by the periodic acid. Rio Hbrtegals silve r carbonate method was attempted to show possible astrocyte proliferation but results were exceedingly poor since the tissue was not fixed i n formalin ammonium bromide. Results and Observations Gross Examination Upon gross examination of the intact rat brains, the most striking change was observed i n the vascular system. About one quarter of the diabetic rats exhibited massive hemorrhages visible on the external surface of the cerebral hemispheres and cerebellum. Several instances of subdural hemorrhage, presumably of venous origin, were noted. It i s assumed that death was the result of these vascular disturbances, especially i n the larger hemorrhages, since death occurred spontaneously i n these animals* One of the control rats had a massive subarachnoid hemorrhage between the cerebrum and cerebellum possibly originating from the Aqueduct of Sulvius. The external appearance of the rat brains and the meninges were normal aside from the vascular disturbances. Upon dissection of the brains, to obtain blocks of tissue suitable for histological embedding, numerous subcortical lesions were observed (FIG. 1). Blood clots associated with the third and l a t e r a l 16 ventricles also were discovered and one instance of a large hemorrhage was found almost completely blocking the brain stem i n the region of the pons« Histological Examination The basic histological change consisted of an alternation i n the walls of the blood vessels culminating i n massive hemorrhage with resulting infarction of the nervous tissue© The vessels most severely affected were the small p i a l arteries and veins with their c o r t i c a l branches. The majority of hemorrhages were of venous origin (FIG. 2) or associated with the ventricles (FIG. 3 and 4) although i n the latter case the vessels of the choroid plexus usually appeared normal. Multiple miliary hemorr-hages of a diffuse nature were very common, occurring i n the cortex, basal nuclei and medulla (FIG. 5)» It i s possible that i n some instances the large area of bleeding i s a result of fusion of these capillary hemorrhages. As a consequence of the pathologic changes of the blood vessels, frequently, Gitter c e l l accumulations were noted i n the perivas-cular space (FIG. 6). The invasions of these macrophagic c e l l s were more frequent i n areas of slight diapedesis rather than massive hemorrhage. The reason for this could be that massive bleeding probably arose spontaneously from the rupturing of a blood vessel and the animals died before neurologic changes could occur. Ar t e r i a l Changes Using Weigert's elastic stain technique i t was possible to show spl i t t i n g and hypertrophy of the elastic fibres i n the larger arteries (FIG. 7)» In the arterioles frequently the entire wall was stained dark purple indicating that elastic hyperplasia was quite extensive. The elastosis i s at f i r s t confined to the intima but eventually i t also invades the media. This i s indicative of the conversion of a muscular type of artery to the elastic type designed to withstand great strain such as present i n hypertension. The amount of elastic tissue i n the walls of an artery apprcacimately corresponds to the pressure of blood within i t . In the affected arteries there was extensive proliferation of the intima and the development of atheromatous areas. In some cases an accumulation or deposition of hyalin material was seen and i n the more severe examples, the lumen was considerably narrowed and sometimes com-pletely obliterated (FIG. 10). Sharply defined nuclei were s t i l l d i s-cernable i n regions of intense hyalin degeneration* Both the hyalin degeneration and the elastic hyperplasia described above are characteristic lesions of arteriosclerosis found i n the benign form of a r t e r i a l hypertension* Other characteristic lesions found were arteriolar necrosis and cellular hyperplasia. Necrosis was observed (FIG. 8) i n the smaller-arteries involving either the whole of the vessel or specific 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 Gitter c e l l invasion, was also observed. Venous changes The majority of the massive,hemorrhages appeared to occur from venous lesions. Although i t was d i f f i c u l t to distinguish necrosis of the cerebral veins due to the thin nature of the walls, quite often hyalin degeneration was noted especially when bleeding was present (FIG. 9). The hyalin 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 into the surrounding nervous tissue of the brain. In treated animals which did not exhibit excessive vascular dysfunction, a frequent observation was of congestion of the cerebral veins, 18 especially those i n the subarachnoid space* It was not possible to ascertain whether the veins were distended beyond their normal diameter but i n some cases the walls appeared extremely thin for the size of the venous lumen* The veins of the salivary glands which were also studied to a lesser degree exhibited the above changes and i n one animal vacuolization was noted i n the media* Fat stain failed 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 salt diet. In summary, the arteriosclerotic and venous changes found i n this study and signifying 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 result of the pathologic changes of the blood vessels of the meninges and cerebral cortex there were circumscribed areas of destruction usually limited to the cortex but occasionally involving the subjacent white matter. Degeneration was also evident i n the medulla and to a slight 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 significant reactions to such vascular injury, fibrous thickening of the connective tissue, was very common. Selective' staining demonstrated this fibrous proliferation to be hyalin i n nature and quite often,association with blood vessels was apparent* However, in 19 some animals these fibrous lesions were i n evidence i n the cbrtex (FIG. 11 and 12) where the only blood vessels were cap i l l a r i e s . Using silver impregnation methods, what appeared to be areas of softening were seen i n a l l parts of the brains of rats showing vascular disturbances. This softening was quite distinct since i t fai l e d to stain and the only structures visible were the capillaries and scant connective tissue elements. In some of the alloxan diabetic animals, large neurons of the medullary nuclei were^degenerated. Shrinkage was observed far i n excess of that due to the fixation and embedding procedure. The cytoplasm became vacuolated considerably and the Nissl granules disappeared (FIG. 14) and i n some cells the nuclei were seen to be necrotic. Sudan IV stain failed 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 tissue, changes were not too evident. In one. treated animal there were distinct areas of the cortex which showed neuron degeneration (FIG. 15). This was especially noticeable i n the polymorphous c e l l layer where the cytoplasm was necrotic and the nuclei swollen and vacuolated. Associated with this cytoplasmic break-down, neuronophagia was seen by the great proliferation of Gitter cells about the c o r t i c a l c e l l s . In summary, the nervous system complications i n the diabetic animals were always associated with concurrent disturbances.of the vascular system. FIG. 1. Spontaneous subarachnoid, hemorrhage on the l e f t w i t h massive cereTaral and sma l l e r hemorrhages on the r i g h t ( Haematoxyl in and e o s i n s t a i n , X 100 ) . FIG. 2 . Spontaneous subarachnoid hemorrhage showing the necrosis of a small vein with resultant "bleeding into the subarachnoid space ( Haematoxylin and eosin stain, X 1 0 0 ) . FIG. 3. Hemorrhage into the lateral ventricle ( haematoxylin and eosin stein, X 120 ). FIG. 4. Hemorrhage from the lateralventricle into the cerebral tissue. The vessels of the choroid plexus appear normal ( Safranin and fast green, X 100 ). FIG. 5. Multiple miliary hemorrhages of the capillaries i n the medulla ( Trichrome stain, X 400 ). FIG. 6. Perivascular accumulation of Gitter cells next to the basal vein and smaller vessels which show some evidence of 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 elastic fibres and hyalinization of the media ( Weigert's resorsin fuchsin stain, X 800). FIG. 8. Two examples of arteries undergoing neorosis. (a) Breakdown of the externa, (b) Breakdown of elastic interna with opening i n wall, also Gitter oell invasion ( Trichrome stain, X 100 ). PIG. 9. Middle oerebral vein with blood in the perivascular spaoe and hyalin i n f i l t r a t i o n into the surrounding tissue ( van Gieson stain, X 200 ). FIG. 10. An oocluded meningeal artery exhibiting extensive intimal proliferation and hyalin i n f i l t r a t i o n ( Trichrome stain, X 1000 ). FIG. 11. Seotion showing fibrous thickening of connective tissue i n cerebral cortex and associated infarotion of the grey matter ( Masson's trichrome stain, X 200 ). FIG. 12. Selective staining i l l u s t r a t i n g hyalin nature of fibrous i n f i l t r a t i o n in cortex ( Van (Jieson stain, X 250 ). FIG. 13. Two areas of softening in the medulla showing only a loose concentration of g l i a l struatures and capillaries remaining ( Modified Cajal silver stain, X 150 ). F I G * 14. Large degenerating motor neurons in the medulla showing loss of Fissl 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 primarily as a histological investigation of the effects of alloxan diabetes on the central nervous system. In reviewing the recent literature 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 fact there i s s t i l l much to be learned about the etiology and pathology of neurological lesions i n diabetes. The striking feature of the results i n this investigation i s the high incidence of vascular damage i n the experimental animals. In the last ten years papers published concerning vascular dysfunction i n diabetes have been extensive and controversial. Dolger (1950) gives a good review of the subject, saying, "The suspected factors i n the development of premature vascular degeneration i n diabetes mellitus have included age, the control of glycosuria and hyperglycemia, the use of insulin, the type of diet, and the excess of cholesterol i n the diet with hypercholesterolemia." The only definite factor established as yet i s the duration of the disease. MLrsky (1946) claims i f the diabetic lives 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 lasted five years or more, free from arteriosclerosis, 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 far exceeds expectation. This i s substantiated by Chute (1948) i n his recent survey. Arteriosclerosis 21 appears inevitablea Opposed to the above views are Hart and Lisa (1944) who admit arteriosclerosis is very common though not consistent in diabetes* They state the combined action of the diabetic and the age factors does not necessarily produce arteriosclerosis even in cases of long duration. Of a more neutral stand is Rabinowitch (1948), who has found arteriosclerotic disease of the heart, arteries and kidneys is 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, is more severe and varies with duration, degree of control and severity of the diabetes. In experimental animals the picture is somewhat different. The most satisfactory method for inducing experimental diabetes is by administration of alloxan. However, this results in Beta -cell necrosis of the islets of Langerhans in 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) in his review on alloxan diabetes states, "It-is of considerable interest that neither diabetic neuritis nor arterios-clerosis has been observed in alloxan diabetic animals." Contrary to this, one year later, Beveridge and Johnson (1950,a) produced cardiovascular and renal lesions in 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 in either group. 22 . Added evidence to Beveridge and Johnson's-work i s obtained through the results of this present investigation. The vascular lesions and cerebral hemorrhage clearly indicate the establishment of arterios-clerosis. Although /the brain i s one of the more common sites for the disease, Thoma ranks the cerebral arteries fourth i n a l i s t , of f i f t e e n commonly elected locations, to the authors knowledge, arteriosclerosis 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 arteriosclerosis. As long ago as 1926, Joslin (1926) thought that a factor i n the premature development of this disease i n diabetes i s an excess of fat i n the diet, i n the body tissues and i n the blood stream. In diabetes the fat and the cholestetol contents of the blood are frequently elevated and this i s compatible with the pathological physiology of arteriosclerosis. It should be remembered, however, that diabetes i s primarily a disturbance of the metabolism of a number of substances and that the exact role of the increased sugar, fat and cho-lesterol contents of the blood awaits a more complete study. In recent years the production of arteriosclerosis i n rabbits i s easily 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© Petruccioli (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 injection of alloxan, concomitant with the i n i t i a l hyperglycemia and showed a strong decrease i n the hypogrlycemic phase. Finall y a l l diabetic animals had an increased blood l i p i d content. Continuing their experiments Beveridge and Johnson (1950,b) discovered the diabetic state had no effect on the level of plasma li p i d s 23 in rats maintained with insulin, with the exception of an increase in total cholesterol* They also noted the presence of the diabetic state did not effect any change in the liver lip i d pattern* • Unfortunately the plasma cholesterol values were unobtainable in this experiment, but i t is presumed that they would be increased in view of other results (Beveridge and Johnson)* This is interesting due to the fact that Pollak (1945) obtained a generalized- atherosclerosis except for the cerebral vessels in cholesterol fed rabbits* When he ligated • both carotid arteries, both jugular<veins or one artery and one vein, lesions of the cerebral vessels developed in both the cholesterol-fed and the control animals* These changes were not;atherosclerotic in nature but hypertensive* 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 hemorr-hage in the brains of alloxanized rats, as found in this experiment defi-nitely indicates a hypertensive condition. These findings are in accord with those of H. W. Radford who carried out a cytological investigation of the endocrine glands of the same experimental animals*. . The changes in the cerebral veins are identical with those noted by Scheinker (1945), who, in studying hypertension associated with massive cerebral hemorrhage, found two types of lesions. The f i r s t was venous congestion, resulting in 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 is considered a terminal phase in 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 fragility as one of. the major causes of brain hemorrhage.* It is interesting to note that hypertensive diabetic patients show a 100$ increase in capillary fragility over the normal and hypertensive non-diabetic patients and diabetic nonhypertensive patients showed a 53$ and 54$ increase respectively (Beaser et al., 1944)* Evidently both diabetes and hypertension are factors predisposing to vascular dysfunctions * . The hypertension observed in these animals is important due to the fact that such a condition has not been noted in diabetic experimental animals to so great a degree before* Since the diabetes is not the inducing factor the only other possibility is the increased salt diet which was fed to both the alloxanized and control animals* This would account for the vascular changes observed in the control rats* It appears therefore that salt-feeding alone can promote hypertension and this condition is 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 is a disturbance in 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 in arterial hypertension and its relation to disturbances in salt balance, however, conclusive results are s t i l l obscure. . „ Possibly the increased salt in the blood promotes an increase in the blood volume with a compensating increase in 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 mellitus but also hypertension i s a result i n the membrane permeability of the ce l l s * It would be interesting to see the results of further work where a close watch was kept on the plasma sodium and chloride content of salt-fed animals to determine the rate of excretion* Quite l i k e l y changes as a result of increased dietary salt for short periods of time are reversibly u n t i l the characteristic vascular lesions have occurred. Nervous system manifestations i n the alloxanized diabetic animals occurred as a result of the disturbances i n the blood supply Jto the brain. 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 condi-tions of the nervous system may be related directly to the - vascular changes that are almost invariably present i n diabetes* The present-day treatment of diabetes has failed 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 to the nerve cells i s due to the anoxia resulting from the disrupted blood supply* The neuron degeneration i n the medulla appears to be the after-effect of peripheral neuropathy, since demyelination occurs i n the extremities and such nerves are connected to motor cells 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 C o n c l u s i o n s T h e c e n t r a l n e r v o u s s y s t e m c o m p l i c a t i o n s o f d i a b e t e s m e l l i t u s a r e s e c o n d a r y t o t h e p a t h o l o g i c a l s t a t e o c c u r r i n g i n t h e v a s c u l a r s y s t e m . I t a p p e a r s t h a t a r t e r i o s c l e r o s i s i s m o r e c o m m o n i n d i a b e t i c a n i m a l s , e i t h e r m a i n t a i n e d o n i n s u l i n o r w i t h u n c o n t r o l l e d d i a b e t e s , t h a n i n n o n - d i a b e t i c r a t s e T h i s m a y b e d u e t o t h e f a c t t h a t d i a b e t e s i s b e l i e v e d t o h a s t e n t h e p r o c e s s e s o f a g i n g , a r t e r i o s c l e r o s i s b e i n g o n e o f t h e m o s t p r e v a l e n t s y m p t o m s . F r o m t h i s e x p e r i m e n t , a n i n c r e a s e d s o d i u m c h l o r i d e d i e t d e f i n i t e l y i n c r e a s e s t h e i n c i d e n c e o f h y p e r t e n s i o n e s p e c i a l l y i n d i a b e t i c a n i m a l s . T h e h i g h i n c i d e n c e o f h e m o r r h a g e i n h y p e r t e n s i v e d i a b e t i c a n i m a l s m i g h t b e d u e t o a n i n c r e a s e i n c a p i l l a r y f r a g i l i t y . 28 Bibliography Bailey, C C , 1949, Vitamins and Hormones, Vol. VII, New York Academic Press Inc., 365 p. Bailey, CC. 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, Capillary F r a g i l i t y i n Relation to Diabetes Mellitus, 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, Brit. J. Exptl. path., 31 : 285* Beveridge, J.M.R. and Johnson, S.E.., 1950, b. Studies i n Diabetic  Rats, Brit. J. Exptl. path., 31 : 294* Boyd, J.D., Jackson, R.L. and Allen, 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  Mellitus, Am. J. Dis. Child., 75 : 1 - 10. Dejong, R., 1950, Nervous System Complications i n Diabetes  Mellitus, J . Nervous & Mental Dis., 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. editor, 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. Int. 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 Islets 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 for the Cause of Diabetes Mellitus, The Harben Lectures of 1949, No. 2, J. Roy. Inst. Pub. Health and Hyg., 13 : 263 - 280. Harris^ W., 1922, Multiple Peripheral Neuritis, Lancet, 2 : 8 4 9 . Hart, J.F. and Lisa, J.R., 1944, Diabetes Mellitus and Arterio- sclerosis, Effect 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 leinroth, L»A •, 1945," Arteriosclerotic Peripheral  Vascular Disease i n Diabetes, Arch. Int. Med., 7 6 : 34 - 38. Hicks, S.P. and Warren, S., 1950> Introduction to Neuropathology, New York, McGraw-Hill Book Co., 1st ed. ~ Joslin, E.P., 1926, Arteriosclerosis and Diabetes, Ann. Clin. Med., 5 : 1061. Jordan, W.R., 1936, Neuritic Manifestations i n Diabetes Mellitus, Arch. Int. Med.j 57 : 307. Jacobs, H.R., - 1937, Hypoglycemic Action of Alloxan, Proc. Soc. Exp. Biol., and Med., 37 :-407. Kaplan, N.O., Franks, M. and Friedgood, C.E., 1945, Metabolism in  Diabetic Coma Produced by Alloxan, Science, 102 : 4 4 7 . Kass, E.H.'and Waisbren, B.A., 1945, A Method for Consistent Induction of Chronic Hyperglycemia with Alloxan, Proc. Soc. Exp.. Bi 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. Lisa, J.R., Magiday, "M. and Hart, J.F., 1942, Peripheral Arterio-. sclerosis 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 for the Future, Diabetes Abst., 5 : 71. Needles, ¥., 1930, Vitamin Studied i n Cases of Diabetic Neuritis, Arch. Neurol, and Psychiat., 41 : 1222. Perera, C-.A. and Blood, D.W., 1946, Hypertension, A Disturbance  i n Salt and Water Metabolism, Am. J. Med.,-1 s 602 - 606o Petruccioli, L., 1949, The Behaviour of the Lipid Content of the  Blood .in the Experimental Alloxan Diabetes, Arch..E» '. ~ Maragliano pat. e Clin., 4 : 705 - 726. Pollak, O.J., 1945, Attempts to Produce Cerebral Atherosclerosis, Arch. Pathology, 39 s 16 - 21. ~ ~ • Rabinowitch,-I.M., 1948, Relationship betvreen Impairment of Liver  Function and Premature Development of Arteriosclerosis i n Diabetes Mellitus, Canad. M. A. <J», 58 : 547 - 556. Randall, L.O., 1938, Changes i n Lipid Composition of Nerves from  Arteriosclerotic and Diabetic patients, J. Bi o l . Chem., 125 : 723. ~~~ ' , Rudy, A« and Epstein, S.H., 1945, Review of loo Cases of Diabetic Neuropathy Followed, from One to 10 years, J. Cl i n . Endocrinol., 5 : 92 - 98. Rundles, R.W.,: 1950, Diabetic Neuropathy, Bull. 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 their 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 Ar 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 Cli n . , 20 : 393. Warren, S., 1938, The Pathology of Diabetes Mellitus, 2nd ed., Philadelphia, Lea and Febriger. 31 YJhite, P.,; 1941, Diabetes i n Youth, New Eng. Jo Medo, 224 : 586. 

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