Experimental diabetic autonomic neuropathy characterization in streptozotocin-diabetic Sprague-Dawley rats

Rats with chronic streptozotocin-induced diabetes develop enlargement of the alimentary tract, loss of fecal consistency, and autonomic neuropathy involving the extrinsic innervation of the ileum. In this study we have continued the characterization of the unmyelinated axonopathy involving the ileal mesenteric nerves of outbred Sprague-Dawley rats diabetic for 1.5 to 10 months using ultrastructural methods. Axonopathy in the alimentary tract of chronically diabetic rats is characterized by modest to marked dilation of axons by distinctive subcellular organelles identical with those described in experimental and clinical axonal dystrophies. Lesions are infrequent 1.5 and 3 months after induction of diabetes, increasing rapidly in numbers thereafter. Axonal lesions are reproducibly encountered in the ileum, cecum, and rectum but not in the proximal jejunum of chronically diabetic rats. Sectioning of mesenteric nerves along their longitudinal axes demonstrates the focal and in some instances apparent terminal nature of the axonal dilation. Disappearance of dystrophic axons distal (peripheral) to mesenteric crush injury confirms the origin of the axons extrinsic to the gut wall. Only rare examples of dystrophic diabetic axonopathy were detected within the wall of the ileum, presumably representing involvement of terminal axonal segments within the intrinsic ganglia. Examination of the mesenteric nerves of 18- and 30-month-old control rats failed to demonstrate dystrophic axonopathy, providing evidence that diabetic axonopathy does not represent premature development of an aging change in mesenteric nerves. Examination of the autonomic innervation of various other tissues including spleen, bladder, vas deferens, and iris, as well as the phrenic, sciatic, vagus, and tail nerves of 4- to 12-month diabetic animals, failed to demonstrate reproducible axonopathy comparable to that involving the alimentary tract of the same animals. The paravascular fascicles of ileal mesenteric nerves of 6- to 7-month diabetic and age-matched control rats were examined by morphometric methods and failed to demonstrate significant loss of axons or an appreciable shift in mean fiber diameter.     

Noradrenergic and cholinergic innervation of the bone marrow

Bone marrow is supplied by sensory and autonomic innervation. Although it is well established that hematopoiesis is regulated by cytokines and cell-to-cell contacts, the role played by neuromediators on the proliferation, differentiation and release of hematopoietic cells is still controversial. We studied the innervation of rat femur bone marrow by means of fluorescence histochemistry and immunohistochemistry. Glyoxylic acid-induced fluorescence was used to demonstrate catecholaminergic nerve fibers. The immunoperoxidase method with nickel amplification was applied to detect the distribution of nerve fibers using antibodies against the general neuronal marker PGP 9.5 (neuron-specific cytoplasmic protein), while the cholinacetyltransferase immunoreactivity was studied by immunohistochemistry. Our results show the presence of an extensive network of innervation in the rat bone marrow, providing a morphological basis for the neural modulation of hemopoiesis.     

Noradrenergic innervation of vasopression-containing neurons in the rat hypothalamic supraoptic nucleus

The noradrenergic innervation of vasopressin (VP)-containing neurons in the supraoptic nucleus (SON) of the rat hypothalamus was studied electron microscopically by using double-labeling immunocytochemistry combining the pre-embedding peroxidase-anti-peroxidase method with post-embedding immunocolloidal gold staining. Noradrenaline-like immunoreactive axon terminals were found to make synaptic contacts with neurophysin II-like immunoreactive neurons in the SON. This study provides morphological evidence for noradrenergic control of neuronal activity of VP-containing neurons at the SON level.     

Sources of autonomic innervation of the appendix

Summary An experimental investigation of the sources of the sympathetic and parasympthetic innervation of the appendix was carried out by Bielschowsky Gros method. The sympathetic innervation is effected by the solar plexus ganglia. Participation of the ventral sacral roots in the parasympathetic innervation is doubtful, whereas participation of the vagus is more probable. Due to the obscurity of morphological results obtained it may only be supposed that the fibers of the cranial and the sacral parasympathetic do not terminate on the cells of the appendix ganglia, but are connected with it through an intermediate link of intraganglionic neurons.(Presented by Active Member AMN SSSR V. N. Ternovskii) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 57, No. 2, No. 113-116, February, 1964     

Noradrenergic and opioidergic alterations in neuropathy in different rat strains

The Fischer 344 (F344) rat strain differs from the Lewis strain in the response to neuropathic pain. Recently, we found that F344 rats totally recover from mechanical allodynia induced by chronic constriction injury (CCI) of the sciatic nerve 28 days after surgery whereas Lewis rats are initiating their recovery at this time point. Thus, the use of this neuropathic pain model in these different rat strains constitutes a good strategy to identify possible target genes involved in the development of neuropathic pain. Since differences between Lewis and F344 rats in their response to pain stimuli in acute pain models have been related to differences in the endogenous opioid and noradrenergic systems, we aimed to determine the levels of expression of key genes of both systems in the spinal cord and dorsal root ganglia (DRG) of both strains 28 days after CCI surgery. Real time RT-PCR revealed minimal changes in gene expression in the spinal cord after CCI despite the strain considered, but marked changes in DRG were observed. A significant upregulation of prodynorphin gene expression occurred only in injured DRG of F344 rats, the most resistant strain to neuropathic pain. In addition, we found a significant downregulation of tyrosine hydroxylase and proenkephalin gene expression levels in both strains whereas delta-opioid receptor was found to be significantly downregulated only in injured DRG of Lewis rats although the same trend was observed in F344 rats. The data strongly suggest that dynorphins could be involved in strain differences concerning CCI resistance.     

Noradrenergic and opioidergic alterations in neuropathy in different rat strains

The Fischer 344 (F344) rat strain differs from the Lewis strain in the response to neuropathic pain. Recently, we found that F344 rats totally recover from mechanical allodynia induced by chronic constriction injury (CCI) of the sciatic nerve 28 days after surgery whereas Lewis rats are initiating their recovery at this time point. Thus, the use of this neuropathic pain model in these different rat strains constitutes a good strategy to identify possible target genes involved in the development of neuropathic pain. Since differences between Lewis and F344 rats in their response to pain stimuli in acute pain models have been related to differences in the endogenous opioid and noradrenergic systems, we aimed to determine the levels of expression of key genes of both systems in the spinal cord and dorsal root ganglia (DRG) of both strains 28 days after CCI surgery. Real time RT-PCR revealed minimal changes in gene expression in the spinal cord after CCI despite the strain considered, but marked changes in DRG were observed. A significant upregulation of prodynorphin gene expression occurred only in injured DRG of F344 rats, the most resistant strain to neuropathic pain. In addition, we found a significant downregulation of tyrosine hydroxylase and proenkephalin gene expression levels in both strains whereas δ-opioid receptor was found to be significantly downregulated only in injured DRG of Lewis rats although the same trend was observed in F344 rats. The data strongly suggest that dynorphins could be involved in strain differences concerning CCI resistance.     

The role of the autonomic innervation in the control of glucagon release during hypoglycaemia in the calf

1. The extent to which the autonomic innervation to the pancreas is implicated in the control of glucagon release during hypoglycaemia has been investigated in calves 3-6 weeks after birth.2. A pronounced rise in plasma glucagon concentration occurred in normal conscious calves in response to hypoglycaemia following administration of insulin (0.1 u./kg). Prior treatment with atropine caused no significant change in the hypoglycaemic response to insulin in these animals but the rise in plasma glucagon concentration was delayed.3. Section of both splanchnic nerves produced no significant change in the tolerance of conscious calves to this small dose of insulin and the changes in plasma glucagon concentration in these animals were within the normal range.4. In contrast, the same dose of insulin produced severe hypoglycaemia, accompanied by convulsions, in atropinized calves with cut splanchnic nerves. In spite of the intensity of the hypoglycaemic stimulus the rise in plasma glucagon concentration was both delayed and diminished in these animals.5. Administration of atropine alone (0.2 mg/kg) to normal fasting calves produced a significant fall in the mean plasma concentrations of both glucose and glucagon (P < 0.01) within 30 min, without affecting that of insulin.6. A significant increase in plasma glucagon concentration also occurred in response to stimulation of the peripheral ends of the thoracic vagi in adrenalectomized calves with cut splanchnic nerves under barbiturate anaesthesia. A rise in mean plasma glucose concentration was also observed in these experiments and found to be significantly correlated with the glucagon response.7. It is concluded that changes in either sympathetic or parasympathetic efferent activity may modify plasma glucagon concentration in the conscious calf, but that only the latter mechanism is likely to be implicated in the response to changes in plasma glucose concentration within the physiological range.     

Simvastatin-induced cardiac autonomic control improvement in fructose-fed female rats

OBJECTIVE:

Because autonomic dysfunction has been found to lead to cardiometabolic disorders and because studies have reported that simvastatin treatment has neuroprotective effects, the objective of the present study was to investigate the effects of simvastatin treatment on cardiovascular and autonomic changes in fructose-fed female rats.

METHODS:

Female Wistar rats were divided into three groups: controls (n = 8), fructose (n = 8), and fructose+simvastatin (n = 8). Fructose overload was induced by supplementing the drinking water with fructose (100 mg/L, 18 wks). Simvastatin treatment (5 mg/kg/day for 2 wks) was performed by gavage. The arterial pressure was recorded using a data acquisition system. Autonomic control was evaluated by pharmacological blockade.

RESULTS:

Fructose overload induced an increase in the fasting blood glucose and triglyceride levels and insulin resistance. The constant rate of glucose disappearance during the insulin intolerance test was reduced in the fructose group (3.4±0.32%/min) relative to that in the control group (4.4±0.29%/min). Fructose+simvastatin rats exhibited increased insulin sensitivity (5.4±0.66%/min). The fructose and fructose+simvastatin groups demonstrated an increase in the mean arterial pressure compared with controls rats (fructose: 124±2 mmHg and fructose+simvastatin: 126±3 mmHg vs. controls: 112±2 mmHg). The sympathetic effect was enhanced in the fructose group (73±7 bpm) compared with that in the control (48±7 bpm) and fructose+simvastatin groups (31±8 bpm). The vagal effect was increased in fructose+simvastatin animals (84±7 bpm) compared with that in control (49±9 bpm) and fructose animals (46±5 bpm).

CONCLUSION:

Simvastatin treatment improved insulin sensitivity and cardiac autonomic control in an experimental model of metabolic syndrome in female rats. These effects were independent of the improvements in the classical plasma lipid profile and of reductions in arterial pressure. These results support the hypothesis that statins reduce the cardiometabolic risk in females with metabolic syndrome.     

Evaluation of autonomic neuropathy in diabetes mellitus

Summary Quantitative assessment of signs or symptoms of neuropathy, and the beat-to-beat variation, valsalva, orthostasis, handgrip and cold pressor tests, and measurements of plasma renin and catecholamine excretion rate were performed in 23 diabetic patients and 10 age-matched normal subjects.Significant inverse correlations were found between the clinical score and the beat-to-beat variation (a test of efferent vagus function) (r=-0.72,P<0.0005) or the pressor response to handgrip (possible test of efferent sympathetic integrity (r=-0.55,P<0.005) or the values of both tests combined (r=-0.79,P«0.0005); but not with the other measured parameters. Beat-to-beat variation was abnormal in all 9 diabetics with increased and in 9 of 14 with normal clinical score, whereas only seven and one patient from these subgroups, respectively, had an abnormal Valsalva ratio. The pressor response to handgrip was only slightly reduced in the diabetic patients, with greater tendency in those with abnormal clincal score. Additional possible indices of adrenergic dysfunction such as the pressor response to cold stimulus, plasma renin levels and noradrenaline or adrenaline excretion rates did not differ significantly between normal subjects and diabetics.These findings demonstrate a greater prevalence of parasympathetic as compared to sympathetic impairment in diabetic autonomic neuropathy; the beat-to-beat variation was the most sensitive among the tests used. An assessment of clinical evidence combined with non-invasive functional procedures such as the beat-to-beat variation and handgrip tests provide a valuable and easy to perform tool in the evaluation of diabetic neuropathy.     

The autonomic innervation of the human greater saphenous vein

The existence of a double catecholaminergic and cholinergic innervation was demonstrated in the human greater saphenous vein. Catecholamine-containing nerve fibres are organized in a network-like plexus localized at the adventitial-medial border. Acetylcholinesterase-containing nerve fibres are arranged in a plexus found at the adventitial-medial border as well. Catecholamine and acetylcholinesterase-containing nerve fibres, while localized in close apposition since they occupy the same portion of the vein, represent two distinct and independent populations of nerve fibres coming likely from the sympathetic and parasympathetic sections of the autonomic nervous system respectively. Our findings demonstrating a close relationship between catecholaminergic and cholinergic nerve fibres within the wall of the human greater saphenous vein offer morphological support to physiological and pharmacological results reported in the literature of a presynaptic control exerted by cholinergic nerves on norepinephrine release at the level of the saphenous vein.     

Sensory and autonomic innervation of the rat eyelid: Neuronal origins and peptide phenotypes

Neuronal origins, peptide phenotypes and target distributions were determined for sensory and autonomic nerves projecting to the eyelid. The retrograde tracer, Fluoro-Ruby, was injected into the superior tarsal muscle and meibomian gland of Sprague-Dawley rats. Labelled neurons were observed within the pterygopalatine (31 ± 6 of a total of 8238 ± 1610 ganglion neurons), trigeminal (173 ± 43 of 62 082 ± 5869) and superior cervical ganglia (184 ± 35 of 21 900 ± 1741). Immunostaining revealed vasoactive intestinal polypeptide immunoreactivity (VIP-ir) in nearly all Fluoro-Ruby-labelled pterygopalatine ganglion neurons (86 ± 5%) but only rarely in trigeminal (0.3 ± 0.3%) or superior cervical (1.4 ± 1.4%) ganglion neurons. Calcitonin gene-related peptide (CGRP)-ir was not observed in pterygopalatine or superior cervical ganglion somata, but was present in 24 ± 4% of trigeminal neurons. Bright dopamine β-hydroxylase (DBH) immunofluorescence was observed in the majority of eyelid-projecting neurons within the superior cervical ganglia (65 ± 5%) and lighter staining was detected in pterygopalatine neurons (63 ± 3%), but no DBH-ir was observed in trigeminal neurons. Examination of eyelid sections revealed dense VIP-ir innervation of meibomian gland acini and vasculature and modest distribution within tarsal muscle. CGRP-ir fibers surrounded ductal and vascular elements of the meibomian gland and the perimeter of tarsal muscle. DBH-ir fibers were associated with meibomian gland blood vessels and acini, and were more densely distributed within tarsal muscle. This study provides evidence for prominent meibomian gland innervation by parasympathetic pterygopalatine ganglion VIP-ir neurons, with more restricted innervation by sensory trigeminal CGRP-ir and sympathetic neurons. Tarsal muscle receives abundant sympathetic innervation, as well as moderate parasympathetic and sensory CGRP-ir projections. The eyelid contains substantial non-CGRP-ir sensory innervation, the targets of which remain undetermined. The distribution of identified autonomic and sensory fibers is consistent with the idea that meibomian gland function, as well as that of the tarsal muscle, is regulated by peripheral innervation.     

Ultrastructural evidence of peripheral neuropathy in spontaneous hyperglycemic Otsuka Long-Evans Tokushima Fatty rats: histopathological similarity to human diabetic neuropathy

The ultrastructure in the peripheral nerves of spontaneous hyperglycemic rats and of two cases of human diabetic neuropathy was compared using electron microscopy. Experimental animals were Otsuka Long-Evans Tokushima Fatty (OLETF) rats, bred to reveal spontaneous hyperglycemia and glucosuria after 20 weeks of age. The ultrastructural findings in the peripheral nerves of OLETF rats consisted of destructive changes of the myelin sheath, followed by axonal degeneration and basal laminal thickening of Schwann cells covering small-caliber axons. These alternations in the endoneurium were associated with the later onset of capillary vasculopathy, which revealed duplication of endothelial basal lamina and proliferation of microvilli at the luminal surface. It was characteristic that these histological alterations were apparently distinguished according to the duration of hyperglycemia between 10 and 30 weeks of age. The two human diabetic cases, who had suffered from non-insulin-dependent diabetes mellitus (NIDDM) for a considerable period, had undergone amputation of the leg following the onset of severe neuropathic ulcerative necrosis. Biopsied specimens from peripheral nerves revealed perineurial thickening, proliferation of endoneurial collagen tissue, loss of myelinated fibers, extension of Schwann cell processes with occasional onion-bulb formation, and endoneurial vascular basal laminal thickening. The results of these comparative histological studies suggested that the alterations occurring in peripheral nerve tissue of experimental diabetic OLETF rats may reflect the early pathological changes of human diabetic neuropathy.     

Day-night variations in urine excretions and hormones in dogs: role of autonomic innervation

Under controlled light-dark cycle (LD 12:12) and two hydratory conditions, mongrel dogs display marked day-night variations in urine flow and in its constituents. Diurnal peaks were found in urine flow and sodium excretion, while nocturnal peaks were observed in osmolality and potassium excretion. Diuretic responses, after fluid intake, were consistently faster and higher during the day than during the night. Nocturnal increases were found in plasma antidiuretic hormone (ADH), in aldosterone and in plasma renin activity (PRA), which could partially explain the present results. In all dogs there was a decrease in ADH level in the early morning hours which could account for the increased urine flow and decreased osmolality observed at this time. Plasma cortisol did not show significant temporal variations throughout the 24-hour period. Kidney denervation did not alter the day-night variations in urinary flow, sodium and osmolality, but affected the pattern of potassium excretion. A neural control of potassium excretion pattern, probably mediated by PRA-aldosterone is tentatively postulated.     

Noradrenergic innervation of the cerebellar cortex in normal and in Purkinje cell degeneration mutant mice: evidence for long term survival following loss of the two major cerebellar cortical neuronal populations

Purkinje cell degeneration mutant mice were examined during the course of Purkinje cell death (26 and 35 days old) and at 3, 5, 9 and 12 months of age. Glyoxylic acid fluorescence histochemistry for catecholamines was used to investigate possible alterations or reorganization of the noradrenergic fibers from the coeruleo-cerebellar system in response to the degeneration of two major cell types in the cerebellar cortex, of which one, the Purkinje cell, is reported to be the major target neuron. In control mice, noradrenergic fibers traveled in linear and tortuous profiles through the granule cell layer, formed pericellular arrays alongside Purkinje cell somata, and branched profusely into both radially oriented and longitudinally oriented chains. The density of noradrenergic varicosities diminished in the molecular layer, there was with age. In the mutants, concomitant with the progressive shrinkage of the molecular layer, there was a progressive increase in the density of noradrenergic varicosities. This was most conspicuous at 9 and 12 months of age, at which time the molecular layer has been depleted not only of Purkinje cell dendrites, but also of parallel fibers. Noradrenergic fibers in these zones formed dense parallel bundles of varicose profiles whose density reached 621.3 +/- 122.8% (mean +/- SD, n = 4) at 9-12 months of age, compared with age-matched controls. Neurochemical measurement of norepinephrine content in whole cerebellum of the Purkinje cell degeneration mutants revealed no change compared with age-matched controls. We conclude that noradrenergic innervation persists in the cerebellar cortex despite the death of Purkinje cells and most of the granule cells. Although we found an increased density of varicosities in the molecular layer of mutant mice, progressing with age, we believe that this can be explained on the basis of the resultant geometry of the altered cerebellar cortex. It appears that the health of the environment surrounding the noradrenergic fibers in cerebellar cortex has little influence on their anatomical integrity.     

Diabetic gastroparesis in association with autonomic neuropathy and microvasculopathy

Gastroparesis is a frequent and sometimes life-threatening complication of diabetes mellitus. Autonomic neuropathy seems to be one of the most important mechanisms underlying this entity, together with the other probable pathologies. The present study was performed in order to identify an alternative to gastric scintigraphy as a screening test. The gastric emptying times of 60 subjects (Group 1: 20 insulin-dependent patients, Group 2: 20 non-insulin-dependent diabetes mellitus patients, and Group 3: 20 healthy volunteers) were monitored by gastric scintigraphy. Perception thresholds for cold, heat, and vibration were tested by a quantitative sensory test, and QTc dispersions were calculated from standard electrocardiography recordings. In addition, fasting blood glucose, hemoglobin A1c and urine beta2-microglobulin and microalbumin concentrations were determined for the patient groups. Funduscopic examination was performed by an independent ophthalmologist. Gastroparesis was determined in both patient groups, regardless of fasting blood glucose and hemoglobin A1c concentrations. A strong correlation was observed between nephropathy, retinopathy, and cardiac autonomic denervation (QTc) and gastroparesis. In conclusion, retinal and renal microvasculopathy parameters and cardiac autonomic function tests may be useful for screening diabetic patients for gastroparesis.