STIMULATION OF NERVES INNERVATING THE DOG'S PANCREAS

 

J. Rozman, 1B. Zorko, 2M. Bunc, 3 M. Žitko

 

 

ITIS d. o. o. Ljubljana, Centre for Implantable Technology and Sensors, Lepi pot 11, University of Ljubljana, 1Veterinary Faculty, Gerbičeva 60,   2 School of Medicine, Institute of Pathophysiology, Zaloška 4, 3University Medical Centre Ljubljana, Department for Nuclear Medicinea, Zaloška 7, 1000 Ljubljana, Republic of Slovenia

 

 

SUMMARY

 

 

Our aim was to modulate secretion of insulin and glucagon into the blood of a healthy and diabetic dog with stimulation of nerves innervating the pancreas. The 33-electrode spiral cuffs were implanted in an adult Beagle canine. The first cuff was installed on the vagus nerve, the second one on the splanchnic nerve and the last one on the pancreatic nerve. To cause insulin-dependent Type I diabetes, partial dysfunction of the pancreas, was induced. Nerves were stimulated using biphasic, rectangular current pulses (10mA, 200ms, 20Hz). Samples from the femoral artery were drawn before the experiment, after 5min and 5min after the stimulation stopped. Results of radioimmunological assay (RIA) of blood samples showed that, in intact pancreas, stimulation of the vagus nerve caused a considerable increase in insulin secretion, not significant change in glucagon secretion. and decrease in C-peptide secretion. Splanchnic nerve stimulation did not cause considerable change in insulin and C-peptide secretion while considerable increse in glucagon secretion was noticed. Pancreatic nerve stimulation did not change considerably the secretion in any of the three hormones. In dysfunctioned pancreas, vagal nerve stimulation caused an increase in insulin and glucagon secretion and minor increase in C-peptide secretion. Splanchnic nerve stimulation caused a minor decrease in insulin secretion, a considerable increase in glucagon secretion. and a small increase in C-peptide secretion. Pancreatic nerve stimulation did not cause considerable change in insulin secretion while minor increase in glucagon and C-peptide secretion was observed.

 

STATE OF THE ART

 

 

In the  field of research considering the possibilities in the application of functional electrical stimulation (FES) of the autonomic nervous system, interest is relatively large. This is evident from the few actually very good but not numerous publications in literature (4, 5). Even less has been done in research involving the selective stimulation of the autonomic nerves as they are, for instance, sympathetic and parasympathetic nerves innervating the pancreas. Since the discovery of insulin in the early twenties (6), research has been concentrated on elucidation of the mechanisms controlling the secretion of hormones by the pancreas (1, 2, 8, 9). Most recent reviews have dealt with the myriad of chemical factors known to affect these hormones, but few have dealt with the great deal of possibilities enabled by the FES of the autonomic peripheral nerves.

 

MATERIALS AND METHODS

 

 

A cuff was made by bonding two 0.1mm thick silicone sheets together. One sheet stretched and fixed in that position was covered by a layer of adhesive. A second unstretched sheet was placed on the adhesive and the composite was compressed. When released, the composite curled into a spiral tube. 33 rectangular platinum electrodes (0.6mm X 1.5mm) connected to the lead wires were mounted on the third silicone sheet. They were arranged in 3 parallel groups each containing 11 electrodes at a distance of 0.5mm. The distance between the spiral groups was 6mm. Accordingly, 11 groups of 3 electrodes in the same line in a longitudinal direction were formed. All electrodes of the central and 2 outer groups were then connected to the lead wires. The sheet with electrodes was then bonded on the inner side of the cuff. The length of the cuff was optimized so that the surface of the nerve covered by the cuff would be as small as possible as shown in Fig. 1.

 


 

 

Fig. 1. 33-electrode spiral cuff for stimulation of nerves innervating the pancreas of a dog.

 

 

Lead wires were connected to the common connector to be implanted within the lateral subcutaneous tissue for the time between experiments. Gas-sterilized cuffs were implanted according to the protocol approved by a local ethics committee. The animal was premedicated with medetomidine 40mg/kg i.m. (Domitor, Orion Corp.) and methadone 0.2mg/kg s.c. (Heptanon, Pliva). Induction was performed with propofol 1.0 to 2.0mg/kg i.v. (Diprivan, Zeneca Pharamaceuticals Ltd.). General anaesthesia was maintained with isoflurane 0.8 to 1.5 vol.% (Forane, Abbott) in 100% O2 (7). Analgesia during surgery was sustained with ketamine 0.5 to 2.0mg/kg i.v. (Ketamine, Veyx-Pharma GmbH) when necessary. Antibiotics (cefazolin 20mg/kg i.v.; Cefamezin, Krka) were administered perioperatively. Analgesia during the early recovery period was provided with methadone 0.3 to 0.5mg/kg s.c. TID. Tramadol 8.0mg/kg s.c. TID (Tramal, Grünenthal GmbH)  was administered for an additional two days. To allow the devices to stabilize, the first experiment was performed 30 days after the implantation. The first cuff was installed on the vagus nerve at the neck as shown in upper part of the Fig. 2. In the splanchnic nerve the cuff was installed on the nerve before the celiac ganglion as shown in the lower part of Fig. 2. In the pancreatic nerve the cuff was installed on the nerve at the site before it enters the pancreas as also shown in the lower part of Fig. 2. To incompletely dysfunction a pancreas, thus causing Type I diabetes, the death of a certain portion of islet b-cells in the pancreas was induced (3). An Alloxan (Sigma Chemical Co., St. Louis, Mo.) was dissolved in a physiological solution (0.9% NaCl) (200mg/ml). A freshly prepared dissolution (50mg/kg) was then intravenously injected into the blood. After 24 hours the pancreas was irreversibly, incompletely dysfunctioned and permanent hyperglycemia was induced. Since the protocols for curing naturally or experimentally induced Type I diabetes are the same insulin (Homofan 100, Pliva) therapy was applied (1IE/kg).

 

 

 

 

Fig. 2. (Upper part) X-ray of implanted 33-electrode spiral cuff showing it’s position on the vagus nerve of a dog; (lower part) X-ray of implanted 33-electrode spiral cuffs showing their position on the splanchnic and pancreatic nerves of a dog.

 

 

To stabilize the blood glucose at a level approximately four times greater than normal a time period of 7 days prior to the stimulation was introduced. Blood samples from the femoral artery were drawn each time before experimental activity to measure the basal level of glucagon, insulin and C-peptide in the blood. They were drawn also at the beginning, after 5min, and 5min after the stimulation stopped.

 

 

RESULTS

 

 

Results of RIA confirmed the hypothesis that, in intact pancreas, stimulation of the vagus nerve caused a considerable increase in insulin secretion (from 13 to almost 110munits/ml). However, vagal nerve stimulation did not cause significant changes in glucagon secretion (from 106 to 125pg/ml). Vagal nerve stimulation caused also some decrease in C-peptide secretion (from 0.74 to 0.34ng/ml). Result also showed that splanchnic nerve stimulation did not cause considerable change in insulin secretion (from 17 to 19munits/ml) while considerable increse in glucagon secretion was noticed (from 125 to 175pg/ml). Splanchnic nerve stimulation did not cause considerable change in C-peptide secretion (from 0.48 to 0.49ng/ml). Pancreatic nerve stimulation did not change considerably the secretion in any of the three hormones. In severed pancreas, vagal nerve stimulation caused an increase in insulin secretion (from 12.6 to 28munits/ml). Moreover, vagal nerve stimulation also increased glucagon secretion (from 119.3 to 130pg/ml). Besides, vagal nerve stimulation caused minor increase in C-peptide secretion (from 0.55 to 0.58ng/l). Splanchnic nerve stimulation caused a minor decrease in insulin secretion (from 15.8 to 13.3munits/ml) and a considerable increase in glucagon secretion was observed (from 74.4 to 133.1 pg/ml). Splanchnic nerve stimulation caused a small increase in C-peptide secretion (from 0.29 to 0.68ng/ml). Pancreatic nerve stimulation did not cause considerable change in insulin secretion (from 6.7 to 6.9munits/ml). while minor increase in glucagon and C-peptide secretion was observed (from 90.9 to 96.1pg/ml for glucagon and from 0.49 to 0.57ng/ml for C-peptide).

 

 

DISCUSSION

 

 

The results could be used in various animal and human basic studies concerning neurophysiology of endocrine glands and internal organs and their relation to bodily changes and disease. Ultimately, the method of FES of peripheral autonomic nerves with multi-electrode spiral cuffs could be used for both stimulation and recording in different combinations. A methodology as well as developed accompanying technological solutions could be used in the transfer of this model to the human alternative model of curing diabetes mellitus. Future studies will focus on chronic, selective stimulation of different superficial regions of nerves innervating the intact and damaged pancreas of a dog. Therefore, the long-range goal of our research will be to understand how the various branches of the utonomic nervous system regulate pancreatic endocrine and exocrine secretion.

 

REFERENCES

 

 

1) Ahrén B. "Autonomic regulation of islet hormone secretion--implications for health and disease", Diabetologia 2000 Apr;43(4):393-410.

2) Ahrén B. "Regulation of insulin secretion by nerves and neuropeptides," Ann Acad Med Singapore 1999 Jan;28(1):99-104

3) Ahrén B, Sundkvist G. "Long-term effects of alloxan in mice", Int J Pancreatol 1995 Apr;17(2):197-201.

4) B. Ahrén and G. J. Taborsky, “The mechanism of vagal nerve stimulation of Glucagon and Insulin secretion in the dog”, Endocrinology, vol. 118, No. 4, pp. 1551-1557, 1986.

5) Ahrén B, Veith RC, Taborsky GJ Jr. "Sympathetic nerve stimulation versus pancreatic norepinephrine infusion in the dog: 1). Effects on basal release of insulin and glucagon", Endocrinology 1987 Jul;121(1):323-31

6) F. G. Banting and S. Gairns, “Factors influencing the production of insulin”, Am. J. Physiol. vol. 68, pp. 24-39, 1924.

7) Havel PJ, Paquette TL, Taborsky GJ Jr. "Halothane is less suppressive than pentobarbital on reflex and neural activation of pancreatic F-cell", Am. J. Physiol. 1986 Jul;251(1 Pt 1):E111-6.

8) M. W. Roy, K. C. Lee, M. S. Jones and R. E. Miller, “Neural control of pancreatic Insulin and somatostain secretion”, Endocrinology, vol. 115, No.2, pp. 770-775, 1984.

9) S. C. Woods and D. Porte, Jr., "Neural control of the endocrine pancreas", Physiological Reviews, vol. 54, No. 3, pp. 596-619, 1987.

 

 

ACKNOWKEDGEMENT-This work was financed by Research Grants: J2-0542 from the Ministry of Education, Science and Sport, Ljubljana, Republic of Slovenia and HPRN-CT-2000-00030 from the European Commission.

 

AUTHOR'S ADDRESS

 

Dr. Janez Rozman, Centre for Implantable Technology and Sensors, Lepi pot 11, 1000 Ljubljana, Republic of Slovena