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0 Microscopic Study of the Intrapancreatic Ganglia and Its effect on the Endocrine 1 and Exocrine Part of Rat pancreas 2 Abstract 3 Background: Pancreas is an important endocrine and exocrine organ which plays a mandatory 4 role in nutritional hemostasis. The intrapancreatic ganglia work like a small brain for rapid 5 control of diet metabolism and any defect in the intrapancreatic ganglia may lead to hormonal 6 imbalance and malfunctioning of the pancreas leading to many disorders like diabetes mellitus. 7 The aim of this study is to identify the morphology and location of the intrapancreatic ganglia 8 and its critical function in controlling both the endocrine and exocrine parts of the pancreas. 9 Methodology: Fourteen rats of both sexes were used in this study. The body weight ranges from 10 250-500gm. The animals were anesthetized by intraperitoneal injections with Sodium 11 Pentobarbital (25mg/Kg). Dissection and histological processing of the pancreas was done for 12 the light microscope examination. 13 Results: The intrapancreatic ganglia were detected as groups of nerve cells and axons in the 14 connective tissue between the pancreatic acini of the rat pancreas. Also, this study reveals that 15 the intrapancreatic ganglia work as a reflex center for regulating any metabolic changes related 16 to the endocrine and exocrine parts of the pancreas. The pathway of the intrapancreatic ganglia is 17 considered a short and direct or indirect pathway with the myenteric plexus in regulating the 18 endocrine and exocrine reaction of the pancreas. However, the degeneration of the 19 intrapancreatic ganglia after vagotomy and sympathectomy was proved and its critical and fast 20 reaction to any metabolic changes was explained by measuring the blood glucose level and 21 glucose tolerance test as well. 22
1 Conclusion: The morphology and location of the intrapancreatic ganglia was identified in the rat 23 pancreas. Also, degenerative changes of intrapancreatic ganglia were observed after denervation 24 and this affect the exocrine and endocrine function of the pancreas (results of previous study). 25 This was approved by a high glucose level both in fasting blood and the glucose tolerance tests, 26 which is consistent with the histological results. Therefore, the intrapancreatic ganglia control 27 any metabolic changes through direct or indirect pathways and any disease that affect the ganglia 28 my affect the exocrine or endocrine function of the pancreas for example it may cause diabetes 29 mellitus. 30 Keywords: Rat pancreas;Intrapancreatic ganglia; Microscopic study;Pathway 31 Corresponding author: 32 Prof.M.H.AL-Muhtaseb.Anatomy Department,Faculty of Medicine,The University of 33 Jordan,Queen Rania str.11942,Amman.Jordan.Phone: 00962777487230 34 E-mail: mhmutseb@ju.edu.jo 35 36 37 38 39 40 41 42
2 43 44 INTRODUCTION 45 Pancreas is an abdominal organ that receives innervation from the higher center (1). Through this 46 innervation and with the effect of hormonal changes, the pancreas plays a major role in 47 nutritional hemostasis (2). The pancreas consists of two major parts, endocrine and exocrine. The 48 endocrine part (Islets of Langerhans) secretes hormones like insulin, glucagon, somatostatin, and 49 polypeptide (3), while the exocrine part releases digestive enzymes (4). There are many 50 similarities in the exocrine and endocrine parts between the rat and human pancreas (5,6,7,8,9) 51 and this make the rats more preferred in our experimental studies. Therefore, the anatomy and 52 histology of the pancreatic region in the rat is important in such studies and can play a major role 53 in satisfying the reliability of the results. 54 The innervation of the pancreas has been known since the 19th century (10). In 1869, 55 Langerhans first described the intrapancreatic ganglia and then many studies were done over 56 many decades (11,12,13,14,15,16,17,18,19). 57 The intrapancreatic ganglia is considered as simple parasympathetic relays, but new evidence 58 suggests that it is similar to the enteric nervous system (20). Intrapancreatic ganglia may 59 constitute a complex information-processing center containing various neurotransmitters and 60 form an endogenous neural network (20,19,21). Although the influence of extra pancreatic 61 nerves, especially autonomic nerves, on pancreatic endocrine function has been extensively 62 studied, the role of intrapancreatic ganglia on endocrine and exocrine functions has less 63 investigations (14,22,20,1). 64
3 Sensory information from the pancreas is sent up to the central nervous system through both 65 vagal and spinal pathways (23,4). Pancreatic vagal afferent (Parasympathetic afferent) neurons 66 transfer sensory information from the pancreas to the brain, these neurons originate in nodose 67 ganglia (24); the central target of these neurons is the nucleus of the solitary tract (25,5). 68 The sympathetic afferent neurons ascend up through dorsal root ganglia T6-L2 (22,1,5); these 69 neurons synapse with interneurons in the spinal cord in laminae I and IV, (26,23,4). 70 On the other hand, nerve input to the pancreas comes from both sympathetic and 71 parasympathetic neurons. The sympathetic innervation of the pancreas originates from the 72 sympathetic preganglionic neurons in the lateral horn of the lower thoracic and upper lumber 73 segments of the spinal cord (27). Axons from these neurons exit in the spinal cord through the 74 ventral roots and supply either the paravertebral ganglia of the sympathetic chain via 75 communicating rami of the thoracic and lumbar nerves or through the celiac and mesenteric 76 ganglia via the splanchnic nerves (2,28,22). These neurons give branches to Islets, blood vessels, 77 ducts, acini, and intrapancreatic ganglia (29,30). 78 Most parasympathetic preganglionic fibers supply the pancreas from the dorsal motor nucleus of 79 the vagus and some may originate from a nucleus ambiguous in the brainstem (22,31,1). The 80 vagus nerve fibers enter the abdomen through esophageal hiatus and give the ventral and dorsal 81 vagal trunk and their branches. Concerning the pancreas, a number of fibers enter directly the 82 pancreas and supply the intrapancreatic ganglia (32). Some of the vagal parasympathetic fibers 83 supply the pancreas indirectly through the myenteric plexus which lies in the duodenum, which 84 in turn sends a number of fibers distributes to the endocrine part (islets of Langerhans), exocrine 85 parts (acini) and intrapancreatic ganglia (2,4). 86
4 In a previous study done by Al-Muhtaseb (33) the effect of denervation on the pancreas result in 87 the degeneration of the endocrine and exocrine part of the rat pancreas and also degeneration of 88 the intrapancreatic ganglia. Measurements of the changes in fasting blood glucose levels after 89 denervation were done and compared with the control group. Both showed significant elevation 90 in blood glucose after denervation. 91 MATERIALS AND METHODS 92 In this study, we used 14 adult albino rats from both sexes which were procured from the Animal 93 House of the Institute. The rats’ body weight ranges from 250-500gm. The animals were 94 anesthetized by intraperitoneal injections with Sodium Pentobarbital (25mg/Kg). All experiments 95 on the animals were conducted according to the NIH guidelines for animal experiments. 96 All animals were sacrificed by perfusion method (33). The pancreas was dissected out, cut into 97 small pieces, and placed in fresh fixatives for 48 hours. Tissues were dehydrated in alcohol, 98 cleared in xylene, and then embedded in paraffin wax. The tissue blocks were cut into 10 μm 99 sections, rehydrated, stained with hematoxylin and eosin, and then examined under the light of 100 the microscope. 101 In the previous study by Al-Muhtaseb (33), the effect of sympathectomy and truncal vagotomy 102 on the rat pancreas was studied on twelve rats. Truncal vagotomy was performed by cutting the 103 anterior and posterior vagal trunks around the esophagus below diaphragm, each trunk was 104 isolated and then a piece of 1 cm was cut and removed between two ligatures. On the other hand, 105 the sympathectomy was performed by cutting the splanchnic nerve fibers projecting to the 106 pancreas, all nerve fibers around the celiac trunk, the right left gastric arteries, and the nerve 107 fibers around the renal arteries (33). 108
5 The twelve rats in each experiment were divided into three subgroups. The first subgroup of 4 109 animals was the control group, the second subgroup of 4 animals were allowed to survive for two 110 weeks after the surgery, while the third subgroup was allowed to survive for three weeks after 111 the surgery (33). The animals which used for surgery were anaesthetized by intraperitoneal 112 injections with sodium pentobarbital (25 mg/kg body weight). 113 Measurements of the fasting blood glucose levels for the rats at different intervals of time after 114 the surgeries were done, in addition to a glucose tolerance test measured two hours after 115 intravenous injection of 0.5 gm/kg body weight of glucose (33). 116 All animals were sacrified by perfusion method through the left ventricle of the heart, first by 117 normal saline, then with 10% formaldehyde fixative. After their death, the pancreas was 118 dissected out and examined under light microscope. 119 RESULTS 120 Microscopic examination of the rats’ pancreas showed that the intrapancreatic ganglia were 121 located in the connective tissue between the pancreatic acini. The ganglia are composed of many 122 neurons and axons. The neurons of the intrapancreatic ganglia vary in size and shape; they can 123 be oval, round, or polygonal in shape. The nerve cells are surrounded by a number of glial cells. 124 The axons of the ganglia are surrounded by Schwan cells which are distributed to all pancreatic 125 effector cells including islets, pancreatic ducts, acini, blood vessels, and other intrapancreatic 126 ganglia (Figures1 and 2). 127 128 The effect of sympathectomy and truncal vagotomy in the study done by Al-Muhtaseb et al. 129 (2020) showed degenerative changes in the intrapancreatic ganglia, the nerve cells showed 130
6 indentation in the plasma membrane, vacuoles in the cytoplasm and changes in the nucleus. The 131 axons and glial cells showed degenerative changes and spaces as well, due to degeneration of the 132 connective tissue seen around the ganglia (figure 3). Also, the glucose tolerance test from the 133 previous study by Al-Muhtaseb et al. (2020) showed a significant increase in blood glucose 134 levels two hours after the intravenous injection of 0.5 gm/kg body weight of glucose in the 135 animals who survived two and three weeks after denervation as a result of the degeneration of 136 the intrapancreatic ganglia (Table 1 and 2). 137 The exocrine portion of the pancreas comprises the bulk of the organ, it is subdivided into 138 lobules by connective tissue septa. Each lobule consists of closely packed secretory acini. The 139 acinus is composed of several pyramidal-shaped cells possessing round nuclei (figure4). 140 The endocrine portion of the pancreas is composed of small spherical clumps of different types 141 of cells – islets of Langerhans, permeated by many fenestrated capillaries which allow quick 142 entry of pancreatic hormones into the blood. The islets of Langerhans are randomly dispersed 143 among the serous acini of the pancreas (figure4). 144 DISCUSSION 145 The distribution of nerves in the pancreas of different animals has already been studied by 146 classical light microscope by using silver impregnation and methylene blue vital-staining 147 methods (34,35,36,37,38,39). Their results provided a fundamental knowledge of the innervation 148 pattern of the pancreas. 149 In this study, the location and correlation between the intrapancreatic ganglia with the endocrine 150 and exocrine parts of the pancreas have been demonstrated. We believe that the pathway of 151 intrapancreatic ganglia for the regulation of endocrine and exocrine secretion in the pancreas, is 152
7 very short, this explain the rapid reflex from the pancreas after each meal or any changes that 153 affect the glucose level and the rapid hemostasis. 154 A study on the mouse pancreas (40), showed that intrapancreatic ganglia contain nerve terminals 155 that form a network of unmyelinated nerve fibers consisting of thin axons associated with the 156 Schwann cell processes., Another study conducted at China Medical University (41), it was 157 found that Intrapancreatic ganglia constitute endogenous neural networks and release various 158 neurotransmitters. They integrate both intrinsic and extrinsic nerve inputs to play an important 159 role in pancreatic endocrine secretion. Both are in agreement with our microscopic findings. 160 Tract-tracing of intrapancreatic ganglionic neurons shows that intrapancreatic ganglia send nerve 161 fibers directly to the islets (42). This ganglion-islet association has been recently demonstrated 162 by a three-dimensional panoramic histological study (21). An electrophysiological study in a 163 ganglion-islet attached preparation setting provides direct evidence for the modulation of insulin 164 secretion by the intrapancreatic ganglia. The activity of the neurons of intrapancreatic ganglionic 165 directly stimulates insulin secretion, while blockage of this activity removes the modulation (14). 166 These results are in agreement with the results of our microscopic findings and ALMuhtaseb 167 study (33). 168 Moreover, several studies have been conducted to investigate the changes in the pancreatic tissue 169 after truncal vagotomy (43). It is well known that the cholinergic parasympathetic postganglionic 170 nerve fibers have a direct effect on the muscarine receptors of the beta cells of the islets of 171 Langerhans, thus stimulating them to release insulin (44). Also, a recent study done by Al-172 Muhtaseb et al. (2020) showed that there are degenerative changes in the intrapancreatic ganglia 173 after denervation. The effect of vagotomy and sympathectomy causes a significant elevation in 174
8 blood glucose levels in the rats’ pancreas. The results of this study suggest that the vagal 175 innervation of the pancreas has a direct and indirect impact on the intrapancreatic ganglia. 176 According to Rossi et al, (2005), the parasympathetic fibers that innervate the endocrine part 177 originate from intrapancreatic ganglia, which are connected with the vagus nerve fibers. 178 In conclusion, intrapancreatic ganglia receive inputs from sympathetic, and parasympathetic 179 fibers and other nerves from the intrapancreatic ganglia. This reveals that the intrapancreatic 180 ganglia control the function of the endocrine and exocrine parts of the pancreas by direct and 181 indirect connection. This is manifested by the morphological changes in the intrapancreatic 182 ganglia and the rise in blood glucose levels after vagotomy and sympathectomy (33). 183 The pattern of innervation of the rats’ pancreas was found very similar to other organs such as 184 the heart. According to Mitchell 1956, sympathetic and parasympathetic nerve fibers join the 185 cardiac plexus around the trachea, then the cardiac plexus innervate the heart directly or 186 indirectly through the intracardiac ganglia (45). Therefore, we can consider the intrapancreatic 187 ganglia as a pacemaker of the pancreas. 188 In the future, more studies are needed to be conducted using histochemistry as that might add 189 more to the findings of the current study. 190 191 192 ACKNOWLEDGEMENTS 193
9 This work was done at the University of Jordan by Afnan Zetawi, an MD, MS degree holder in 194 Anatomy and Histology – School of Medicine under the supervision of Professor M.H.AL-195 Muhtaseb. 196 197 198 References: 199 (1) Rodriguez-Diaz R, Caicedo A. Neural control of the endocrine pancreas. Best practice & 200 research Clinical endocrinology & metabolism. 2014 Oct 1;28(5):745-56. 201 (2) Ahrén B. Autonomic regulation of islet hormone secretion–implications for health and 202 disease. Diabetologia. 2000 Apr;43:393-410. 203 (3) Rossi J, Santamäki P, Airaksinen MS, Herzig KH. Parasympathetic innervation and function 204 of endocrine pancreas requires the glial cell line–derived factor family receptor α2 (GFRα2). 205 Diabetes. 2005 May 1;54(5):1324-30. 206 (4) Babic T, Travagli RA. Neural control of the pancreas. Pancreapedia: The Exocrine Pancreas 207 Knowledge Base. 2016 Sep 23. 208 (5) Dolenšek J, Rupnik MS, Stožer A. Structural similarities and differences between the human 209 and the mouse pancreas. Islets. 2015 Jan 2;7(1):e1024405. 210 (6) Tiscornia OM. The neural control of exocrine and endocrine pancreas. The American journal 211 of gastroenterology. 1977 Jun;67(6):541-60. 212
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11 (16) Love JA, Szebeni K. Morphology and histochemistry of the rabbit pancreatic innervation. 234 Pancreas. 1999 Jan 1;18(1):53-64. 235 (17) Sha L, Szurszewski JH. Leptin modulates fast synaptic transmission in dog pancreatic 236 ganglia. Neuroscience letters. 1999 Mar 26;263(2-3):93-6. 237 (18) Yi E, Smith TG, Love JA. Noradrenergic innervation of rabbit pancreatic ganglia. 238 Autonomic Neuroscience. 2005 Feb 7;117(2):87-96. 239 (19) Shen Q, Wang Y, Zhang N, Gao D, Liu Y, Sha L. Substance P expresses in intrapancreatic 240 ganglia of the rats. Neuropeptides. 2016 Oct 1;59:33-8. 241 (20) Gylfe E, Tengholm A. Neurotransmitter control of islet hormone pulsatility. Diabetes, 242 Obesity and Metabolism. 2014 Sep;16(Suppl 1):102-10. 243 (21) Tang SC, Shen CN, Lin PY, Peng SJ, Chien HJ, Chou YH, Chamberlain CE, Pasricha PJ. 244 Pancreatic neuro-insular network in young mice revealed by 3D panoramic histology. 245 Diabetologia. 2018 Jan;61:158-67. 246 (22) Love JA, Yi E, Smith TG. Autonomic pathways regulating pancreatic exocrine secretion. 247 Autonomic Neuroscience. 2007 Apr 30;133(1):19-34. 248 (23) Ahren B, Ericson LE, Lundquist I, Loren I, Sundler F. Adrenergic innervation of pancreatic 249 islets and modulation of insulin secretion by the sympatho-adrenal system. Cell and tissue 250 research. 1981 Mar;216:15-30. 251 (24) de Lartigue G. Role of the vagus nerve in the development and treatment of diet‐induced 252 obesity. The Journal of physiology. 2016 Oct 15;594(20):5791-815. 253
12 (25) Renehan WE, Zhang XU, Beierwaltes WH, Fogel RO. Neurons in the dorsal motor nucleus 254 of the vagus may integrate vagal and spinal information from the GI tract. American Journal of 255 Physiology-Gastrointestinal and Liver Physiology. 1995 May 1;268(5):G780-90. 256 (26) Brunicardi FC, Shavelle DM, Andersen DK. Neural regulation of the endocrine pancreas. 257 International journal of pancreatology. 1995 Dec;18:177-95. 258 (27) Llewellyn-Smith IJ. Anatomy of synaptic circuits controlling the activity of sympathetic 259 preganglionic neurons. Journal of chemical neuroanatomy. 2009 Nov 1;38(3):231-9. 260 (28) Gilon P, Henquin JC. Mechanisms and physiological significance of the cholinergic control 261 of pancreatic β-cell function. Endocrine reviews. 2001 Oct 1;22(5):565-604. 262 (29) Yi SQ, Miwa K, Ohta T, Kayahara M, Kitagawa H, Tanaka A, Shimokawa T, Akita K, 263 Tanaka S. Innervation of the pancreas from the perspective of perineural invasion of pancreatic 264 cancer. Pancreas. 2003 Oct 1;27(3):225-9. 265 (30) Havel PJ, Taborsky Jr GJ. The contribution of the autonomic nervous system to changes of 266 glucagon and insulin secretion during hypoglycemic stress. Endocrine reviews. 1989 Aug 267 1;10(3):332-50. 268 (31) Chandra R, Liddle RA. Modulation of pancreatic exocrine and endocrine secretion. Current 269 opinion in gastroenterology. 2013 Sep;29(5):517. 270 (32) Chandra R, Liddle RA. Neural and hormonal regulation of pancreatic secretion. Current 271 opinion in gastroenterology. 2009 Sep;25(5):441. 272 (33) Al-Muhtaseb MH, Mohtasib HM, Khouri TR. Microscopic study of the rat pancreas after 273 denervation. Eur. j. anat. 2020;24(1):01-7. 274
13 (34) Cajal SR. Les nouvelles idées sur la structure du système nerveux chez l’homme et chez les 275 vertébrés. C. Reinwald & Cie, libraires-éditeurs; 1894. 276 (35) Cajal SR. Terminacion de los nervios y tubos glandulares del pancreas de los vertebrados. 277 Trab. Lab. Histol. Faculd. Med. Barcelona. 1981. 278 (36) Castro FD. Contribution à la connaissance de l’innervation du pancréas. Trav. Lab. Rech. 279 Biol. Univ. Madrid. 1923;21:423-57. 280 (37) Honjin R. The innervation of the pancreas of the mouse, with special reference to the 281 structure of the peripheral extension of the vegetative nervous system. Journal of Comparative 282 Neurology. 1956 Jun;104(3):331-71. 283 (38) Pensa A. Osservazioni sulla distribuzione dei vasi sanguigni e dei nervi nel pancreas. Int. 284 Monatsschr. Anat. Physiol.. 1905;22:90-125. 285 (39) Pines L, Tropowa M. Zur Innervation des Pankreas. Z. Mikrosk. Anat. Forsch.. 1930;20:20-286 50. 287 (40) Ushiki T, Watanabe S. Distribution and ultrastructure of the autonomic nerves in the mouse 288 pancreas. Microscopy research and technique. 1997 Jun 1;37(5‐6):399-406. 289 (41) Li W, Yu G, Liu Y, Sha L. Intrapancreatic ganglia and neural regulation of pancreatic 290 endocrine secretion. Frontiers in neuroscience. 2019 Feb 20;13:21. 291 (42) Kirchgessner AL, Pintar JE. Guinea pig pancreatic ganglia: Projections, transmitter content, 292 and the type‐specific localization of monoamine oxidase. Journal of comparative neurology. 293 1991 Mar 22;305(4):613-31. 294
14 (43) Wormsley KG. The effect of vagotomy on the human pancreatic response to direct and 295 indirect stimulation. Scandinavian Journal of Gastroenterology. 1972 Jan 1;7(1):85-91. 296 (44) Henquin JC, Nenquin M. The muscarinic receptor subtype in mouse pancreatic B-cells. 297 FEBS letters. 1988 Aug 15;236(1):89-92. 298 (45) Mitchell GA. Cardiovascular innervation. (No Title). 1956. E7S Livingstone LTD, 299 Edinburgh and London, pp 238-267. 300 301 302 Tables: 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 Control 2 weeks 3 weeks After Truncal Vagotomy 3.3-4.4 mmol/L 6.3-8.5 mmol/L 8.5-10.2 mmol/L After Sympathectomy 3.5-4.5 mmol/L 6.5-8.8 mmol/L 8.7-10.5 mmol/L Table 1: Fasting Blood Glucose levels in mmol/L Control 2 weeks 3 weeks After Truncal Vagotomy 4.2-4.9 mmol/L 8.9-11.2 mmol/L 10.4-14.2 mmol/L After Sympathectomy 4.0-4.5 mmol/L 8.4-10.6 mmol/L 8.9-12.6 mmol/L Table 2: Glucose Tolerance Test levels in mmol/L
15 Figures: 319 320 Figure (1): Light micrograph of a section in rat pancreas showing the intrapancreatic ganglia 321 contains nerve cell nucleus (N), axons (A), Schwan cell (Sch) (H&Ex400). 322
16 323 Figure (2): inside the square, intrapancreatic ganglia lies in connective tissue between the 324 pancreatic acini, contains nerve cell (N), schwan cell (Sch) and axons (A) (H&Ex400). 325 326 327
17 Figure (3): inside the square, degenerative changes in the intrapancreatic ganglia and spaces in 328 the connective tissue after sympathectomy and vagotomy, nerve cell (N), schwan cell (Sch) and 329 axons (A) (H&Ex200). 330 331 332 333 Figure (4): Light micrograph of a section in the rat pancreas showing the endocrine and exocrine 334 cells (H&Ex200). 335
0 Microscopic Study of the Intrapancreatic Ganglia and Its effect on the Endocrine 1 and Exocrine Part of Rat pancreas 2 Abstract 3 Background: Pancreas is an important endocrine and exocrine organ which plays a mandatory 4 role in nutritional hemostasis. The intrapancreatic ganglia work like a small brain for rapid 5 control of diet metabolism and any defect in the intrapancreatic ganglia may lead to hormonal 6 imbalance and malfunctioning of the pancreas leading to many disorders like diabetes mellitus. 7 The aim of this study is to identify the morphology and location of the intrapancreatic ganglia 8 and its critical function in controlling both the endocrine and exocrine parts of the pancreas. 9 Methodology: Fourteen rats of both sexes were used in this study. The body weight ranges from 10 250-500gm. The animals were anesthetized by intraperitoneal injections with Sodium 11 Pentobarbital (25mg/Kg). Dissection and histological processing of the pancreas was done for 12 the light microscope examination. 13 Results: The intrapancreatic ganglia were detected as groups of nerve cells and axons in the 14 connective tissue between the pancreatic acini of the rat pancreas. Also, this study reveals that 15 the intrapancreatic ganglia work as a reflex center for regulating any metabolic changes related 16 to the endocrine and exocrine parts of the pancreas. The pathway of the intrapancreatic ganglia is 17 considered a short and direct or indirect pathway with the myenteric plexus in regulating the 18 endocrine and exocrine reaction of the pancreas. However, the degeneration of the 19 intrapancreatic ganglia after vagotomy and sympathectomy was proved and its critical and fast 20 reaction to any metabolic changes was explained by measuring the blood glucose level and 21 glucose tolerance test as well. 22
1 Conclusion: The morphology and location of the intrapancreatic ganglia was identified in the rat 23 pancreas. Also, degenerative changes of intrapancreatic ganglia were observed after denervation 24 and this affect the exocrine and endocrine function of the pancreas (results of previous study). 25 This was approved by a high glucose level both in fasting blood and the glucose tolerance tests, 26 which is consistent with the histological results. Therefore, the intrapancreatic ganglia control 27 any metabolic changes through direct or indirect pathways and any disease that affect the ganglia 28 my affect the exocrine or endocrine function of the pancreas for example it may cause diabetes 29 mellitus. 30 Keywords: Rat pancreas;Intrapancreatic ganglia; Microscopic study;Pathway 31 Corresponding author: 32 Prof.M.H.AL-Muhtaseb.Anatomy Department,Faculty of Medicine,The University of 33 Jordan,Queen Rania str.11942,Amman.Jordan.Phone: 00962777487230 34 E-mail: mhmutseb@ju.edu.jo 35 36 37 38 39 40 41 42
2 43 44 INTRODUCTION 45 Pancreas is an abdominal organ that receives innervation from the higher center (1). Through this 46 innervation and with the effect of hormonal changes, the pancreas plays a major role in 47 nutritional hemostasis (2). The pancreas consists of two major parts, endocrine and exocrine. The 48 endocrine part (Islets of Langerhans) secretes hormones like insulin, glucagon, somatostatin, and 49 polypeptide (3), while the exocrine part releases digestive enzymes (4). There are many 50 similarities in the exocrine and endocrine parts between the rat and human pancreas (5,6,7,8,9) 51 and this make the rats more preferred in our experimental studies. Therefore, the anatomy and 52 histology of the pancreatic region in the rat is important in such studies and can play a major role 53 in satisfying the reliability of the results. 54 The innervation of the pancreas has been known since the 19th century (10). In 1869, 55 Langerhans first described the intrapancreatic ganglia and then many studies were done over 56 many decades (11,12,13,14,15,16,17,18,19). 57 The intrapancreatic ganglia is considered as simple parasympathetic relays, but new evidence 58 suggests that it is similar to the enteric nervous system (20). Intrapancreatic ganglia may 59 constitute a complex information-processing center containing various neurotransmitters and 60 form an endogenous neural network (20,19,21). Although the influence of extra pancreatic 61 nerves, especially autonomic nerves, on pancreatic endocrine function has been extensively 62 studied, the role of intrapancreatic ganglia on endocrine and exocrine functions has less 63 investigations (14,22,20,1). 64
3 Sensory information from the pancreas is sent up to the central nervous system through both 65 vagal and spinal pathways (23,4). Pancreatic vagal afferent (Parasympathetic afferent) neurons 66 transfer sensory information from the pancreas to the brain, these neurons originate in nodose 67 ganglia (24); the central target of these neurons is the nucleus of the solitary tract (25,5). 68 The sympathetic afferent neurons ascend up through dorsal root ganglia T6-L2 (22,1,5); these 69 neurons synapse with interneurons in the spinal cord in laminae I and IV, (26,23,4). 70 On the other hand, nerve input to the pancreas comes from both sympathetic and 71 parasympathetic neurons. The sympathetic innervation of the pancreas originates from the 72 sympathetic preganglionic neurons in the lateral horn of the lower thoracic and upper lumber 73 segments of the spinal cord (27). Axons from these neurons exit in the spinal cord through the 74 ventral roots and supply either the paravertebral ganglia of the sympathetic chain via 75 communicating rami of the thoracic and lumbar nerves or through the celiac and mesenteric 76 ganglia via the splanchnic nerves (2,28,22). These neurons give branches to Islets, blood vessels, 77 ducts, acini, and intrapancreatic ganglia (29,30). 78 Most parasympathetic preganglionic fibers supply the pancreas from the dorsal motor nucleus of 79 the vagus and some may originate from a nucleus ambiguous in the brainstem (22,31,1). The 80 vagus nerve fibers enter the abdomen through esophageal hiatus and give the ventral and dorsal 81 vagal trunk and their branches. Concerning the pancreas, a number of fibers enter directly the 82 pancreas and supply the intrapancreatic ganglia (32). Some of the vagal parasympathetic fibers 83 supply the pancreas indirectly through the myenteric plexus which lies in the duodenum, which 84 in turn sends a number of fibers distributes to the endocrine part (islets of Langerhans), exocrine 85 parts (acini) and intrapancreatic ganglia (2,4). 86
4 In a previous study done by Al-Muhtaseb (33) the effect of denervation on the pancreas result in 87 the degeneration of the endocrine and exocrine part of the rat pancreas and also degeneration of 88 the intrapancreatic ganglia. Measurements of the changes in fasting blood glucose levels after 89 denervation were done and compared with the control group. Both showed significant elevation 90 in blood glucose after denervation. 91 MATERIALS AND METHODS 92 In this study, we used 14 adult albino rats from both sexes which were procured from the Animal 93 House of the Institute. The rats’ body weight ranges from 250-500gm. The animals were 94 anesthetized by intraperitoneal injections with Sodium Pentobarbital (25mg/Kg). All experiments 95 on the animals were conducted according to the NIH guidelines for animal experiments. 96 All animals were sacrificed by perfusion method (33). The pancreas was dissected out, cut into 97 small pieces, and placed in fresh fixatives for 48 hours. Tissues were dehydrated in alcohol, 98 cleared in xylene, and then embedded in paraffin wax. The tissue blocks were cut into 10 μm 99 sections, rehydrated, stained with hematoxylin and eosin, and then examined under the light of 100 the microscope. 101 In the previous study by Al-Muhtaseb (33), the effect of sympathectomy and truncal vagotomy 102 on the rat pancreas was studied on twelve rats. Truncal vagotomy was performed by cutting the 103 anterior and posterior vagal trunks around the esophagus below diaphragm, each trunk was 104 isolated and then a piece of 1 cm was cut and removed between two ligatures. On the other hand, 105 the sympathectomy was performed by cutting the splanchnic nerve fibers projecting to the 106 pancreas, all nerve fibers around the celiac trunk, the right left gastric arteries, and the nerve 107 fibers around the renal arteries (33). 108
5 The twelve rats in each experiment were divided into three subgroups. The first subgroup of 4 109 animals was the control group, the second subgroup of 4 animals were allowed to survive for two 110 weeks after the surgery, while the third subgroup was allowed to survive for three weeks after 111 the surgery (33). The animals which used for surgery were anaesthetized by intraperitoneal 112 injections with sodium pentobarbital (25 mg/kg body weight). 113 Measurements of the fasting blood glucose levels for the rats at different intervals of time after 114 the surgeries were done, in addition to a glucose tolerance test measured two hours after 115 intravenous injection of 0.5 gm/kg body weight of glucose (33). 116 All animals were sacrified by perfusion method through the left ventricle of the heart, first by 117 normal saline, then with 10% formaldehyde fixative. After their death, the pancreas was 118 dissected out and examined under light microscope. 119 RESULTS 120 Microscopic examination of the rats’ pancreas showed that the intrapancreatic ganglia were 121 located in the connective tissue between the pancreatic acini. The ganglia are composed of many 122 neurons and axons. The neurons of the intrapancreatic ganglia vary in size and shape; they can 123 be oval, round, or polygonal in shape. The nerve cells are surrounded by a number of glial cells. 124 The axons of the ganglia are surrounded by Schwan cells which are distributed to all pancreatic 125 effector cells including islets, pancreatic ducts, acini, blood vessels, and other intrapancreatic 126 ganglia (Figures1 and 2). 127 128 The effect of sympathectomy and truncal vagotomy in the study done by Al-Muhtaseb et al. 129 (2020) showed degenerative changes in the intrapancreatic ganglia, the nerve cells showed 130
6 indentation in the plasma membrane, vacuoles in the cytoplasm and changes in the nucleus. The 131 axons and glial cells showed degenerative changes and spaces as well, due to degeneration of the 132 connective tissue seen around the ganglia (figure 3). Also, the glucose tolerance test from the 133 previous study by Al-Muhtaseb et al. (2020) showed a significant increase in blood glucose 134 levels two hours after the intravenous injection of 0.5 gm/kg body weight of glucose in the 135 animals who survived two and three weeks after denervation as a result of the degeneration of 136 the intrapancreatic ganglia (Table 1 and 2). 137 The exocrine portion of the pancreas comprises the bulk of the organ, it is subdivided into 138 lobules by connective tissue septa. Each lobule consists of closely packed secretory acini. The 139 acinus is composed of several pyramidal-shaped cells possessing round nuclei (figure4). 140 The endocrine portion of the pancreas is composed of small spherical clumps of different types 141 of cells – islets of Langerhans, permeated by many fenestrated capillaries which allow quick 142 entry of pancreatic hormones into the blood. The islets of Langerhans are randomly dispersed 143 among the serous acini of the pancreas (figure4). 144 DISCUSSION 145 The distribution of nerves in the pancreas of different animals has already been studied by 146 classical light microscope by using silver impregnation and methylene blue vital-staining 147 methods (34,35,36,37,38,39). Their results provided a fundamental knowledge of the innervation 148 pattern of the pancreas. 149 In this study, the location and correlation between the intrapancreatic ganglia with the endocrine 150 and exocrine parts of the pancreas have been demonstrated. We believe that the pathway of 151 intrapancreatic ganglia for the regulation of endocrine and exocrine secretion in the pancreas, is 152
7 very short, this explain the rapid reflex from the pancreas after each meal or any changes that 153 affect the glucose level and the rapid hemostasis. 154 A study on the mouse pancreas (40), showed that intrapancreatic ganglia contain nerve terminals 155 that form a network of unmyelinated nerve fibers consisting of thin axons associated with the 156 Schwann cell processes., Another study conducted at China Medical University (41), it was 157 found that Intrapancreatic ganglia constitute endogenous neural networks and release various 158 neurotransmitters. They integrate both intrinsic and extrinsic nerve inputs to play an important 159 role in pancreatic endocrine secretion. Both are in agreement with our microscopic findings. 160 Tract-tracing of intrapancreatic ganglionic neurons shows that intrapancreatic ganglia send nerve 161 fibers directly to the islets (42). This ganglion-islet association has been recently demonstrated 162 by a three-dimensional panoramic histological study (21). An electrophysiological study in a 163 ganglion-islet attached preparation setting provides direct evidence for the modulation of insulin 164 secretion by the intrapancreatic ganglia. The activity of the neurons of intrapancreatic ganglionic 165 directly stimulates insulin secretion, while blockage of this activity removes the modulation (14). 166 These results are in agreement with the results of our microscopic findings and ALMuhtaseb 167 study (33). 168 Moreover, several studies have been conducted to investigate the changes in the pancreatic tissue 169 after truncal vagotomy (43). It is well known that the cholinergic parasympathetic postganglionic 170 nerve fibers have a direct effect on the muscarine receptors of the beta cells of the islets of 171 Langerhans, thus stimulating them to release insulin (44). Also, a recent study done by Al-172 Muhtaseb et al. (2020) showed that there are degenerative changes in the intrapancreatic ganglia 173 after denervation. The effect of vagotomy and sympathectomy causes a significant elevation in 174
8 blood glucose levels in the rats’ pancreas. The results of this study suggest that the vagal 175 innervation of the pancreas has a direct and indirect impact on the intrapancreatic ganglia. 176 According to Rossi et al, (2005), the parasympathetic fibers that innervate the endocrine part 177 originate from intrapancreatic ganglia, which are connected with the vagus nerve fibers. 178 In conclusion, intrapancreatic ganglia receive inputs from sympathetic, and parasympathetic 179 fibers and other nerves from the intrapancreatic ganglia. This reveals that the intrapancreatic 180 ganglia control the function of the endocrine and exocrine parts of the pancreas by direct and 181 indirect connection. This is manifested by the morphological changes in the intrapancreatic 182 ganglia and the rise in blood glucose levels after vagotomy and sympathectomy (33). 183 The pattern of innervation of the rats’ pancreas was found very similar to other organs such as 184 the heart. According to Mitchell 1956, sympathetic and parasympathetic nerve fibers join the 185 cardiac plexus around the trachea, then the cardiac plexus innervate the heart directly or 186 indirectly through the intracardiac ganglia (45). Therefore, we can consider the intrapancreatic 187 ganglia as a pacemaker of the pancreas. 188 In the future, more studies are needed to be conducted using histochemistry as that might add 189 more to the findings of the current study. 190 191 192 ACKNOWLEDGEMENTS 193
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15 Figures: 319 320 Figure (1): Light micrograph of a section in rat pancreas showing the intrapancreatic ganglia 321 contains nerve cell nucleus (N), axons (A), Schwan cell (Sch) (H&Ex400). 322
16 323 Figure (2): inside the square, intrapancreatic ganglia lies in connective tissue between the 324 pancreatic acini, contains nerve cell (N), schwan cell (Sch) and axons (A) (H&Ex400). 325 326 327
17 Figure (3): inside the square, degenerative changes in the intrapancreatic ganglia and spaces in 328 the connective tissue after sympathectomy and vagotomy, nerve cell (N), schwan cell (Sch) and 329 axons (A) (H&Ex200). 330 331 332 333 Figure (4): Light micrograph of a section in the rat pancreas showing the endocrine and exocrine 334 cells (H&Ex200). 335