In vitro study of acute elevations of endoneurial pressure in mammalian peripheral nerve sheath

In previous studies of peripheral nerve edema, endoneurial fluid pressure rarely exceeded 7 mm Hg for even the most edematous nerves. In a recent study a time-dependent reduction in elastic modulus was reported. However, because of the chronic nature of the studies, pressure and volume changes were not directly recorded from the same nerves. In the present study mammaliam perineurial compliance was directly studied in vitro. When compliance ($\text{Δ}\text{V}\text{Δ}\text{P}$) was compared between nerves subjected to slow rates of ΔV (0.7μl/min) and fast rates (7 μl/min), mean values were reduced for all pressure intervals but did not become statistically significant because of considerable variability between nerves. However, in a separate series of experiments, when the same nerve sheath was consecutively subjected to these two rates of Δ V, compliance was consistently and significantly (P < 0.001, paired t test) greater for slow ΔV, confirming directly the importance of a time-dependent change in elastic modulus, i.e., a viscous modulus. A tentative physiologic-morphologic correlation is made. No evidence was found for norepinephrine- or acetylcholine-responsive contractile elements.     

Degenerative neuropathy in insulin-treated diabetic rats

Peripheral neuropathic alterations associated with diabetes and its treatment with insulin were studied in alloxan-induced diabetic rats. Treatment regimens included daily injections of Protamine Zinc Insulin (PZI), daily injections of Ultralente Insulin and subcutaneously implanted osmotic minipump delivered insulin. Non-diabetic and untreated diabetic groups served as controls. Two separate but similar studies were run, one lasting 4 weeks and the other 8 weeks. Conduction velocities performed on both sensory and motor nerves revealed no statistically significant differences among groups. Anatomical analysis of teased fibers from tibial nerves showed a significant number of fibers with ovoids, consistent with Wallerian-type axonal degeneration, only in the treated diabetic groups. Degeneration was especially severe in the PZI-treated group. Metabolic studies were performed using incorporation of radioactive isotopes ([³H]fucose, [¹⁴C]leucine) into myelin proteins of sciatic nerves. The ratio of $\text{[}{}^3\text{H]fucose}\text{[}{}^{14}\text{C]leucine}$ for the PZI-treated group was significantly decreased when compared to the control groups in both the 4 and 8 week study whereas the minipump-treated group showed no statistically significant difference from the control group in either study. Similar decreases in this ratio have been seen in conditions of peripheral nerve degeneration. It is concluded that daily injections of PZI insulin result in significant nerve degeneration in the alloxan diabetic rat, while continuous levels of insulin delivered by osmotic minipumps result in less degeneration.     

Effects of NaCl and sultopride on striatal [3H]spiperone binding in neonatal,adult and senescent rats

Effects of NaCl, (+)-and (?)-sultopride on striatal [³H]spiperone binding was investigated in 7-day, 70-day and 2-year-old rats. The amount of specific [³H]spiperone binding was the highest at 70 days and the value at adult stage was significantly (P < 0.001) higher than those at 7 days and 2 years. NaCl (100 mM) significantly increased [³H]spiperone binding in neonatal (P < 0.01), adult (P < 0.05) and senescent (P < 0.05) animals. Scatchard analysis showed that the Bmax of low-affinity [³H]spiperone binding was significantly elevated by 100 mM NaCl compared to the value in control of adult animals. More potent inhibition of (?)-sultopride for [³H]spiperone binding than that of the (+)-enantiomer at adult stage was also observed at neonatal and senescent stages. NaCl (100 mM) significantly enhanced inhibitory activities of (+)- and (?)-sultopride at every stage. It is suggested that stabilizing effect of Na⁺ on dopamine (DA) receptor complexes and increasing effect of Na⁺ on binding affinity of benzamide to DA₂ receptors keep functions through development and aging.     

Insulin and the insulin-like growth factors I and II are mitogenic to cultured rat sciatic nerve segments and stimulate [3H]thymidine incorporation through their respective receptors

The factors that control proliferation of Schwann cells during peripheral nerve regeneration are not yet known. In this study we investigated the effects of insulin, insulin-like growth factor I and II (IGF-I and IGF-II), IGF-I analogues, and factors that interfere with their respective receptors, on [³H]thymidine incorporation into cultured nerve segments from the rat sciatic nerve. Segments cultured in nM (0.1–1.7 nM) concentrations of insulin, truncated IGF-I (tIGF-I), long R³IGF-I, or IGF-II exhibited an increase in [³H]thymidine incorporation compared with control segments. IGF-II was most potent. JB1, an IGF-I antagonist, counteracted the effects of tIGF-I and insulin. The results suggest that non-neuronal cells in the nerve segment, probably Schwann cells, possess distinct receptors for insulin, IGF-I, and IGF-II and that these receptors may be involved in the control of Schwann cell proliferation during peripheral nerve regeneration. © 1996 Wiley-Liss, Inc.     

Neuronal stimulation of [3H]thymidine incorporation by primary cultures of highly purified non-neuronal cells

A specific intercellular interaction has been demonstrated between neuronal and non-neuronal cells that appears to increase the rate of non-neuronal cell proliferation. Isolated and recombined primary cultures of both cell types were prepared from 11-day embryonic chick sympathetic ganglia by a method recently developed in this laboratory. When non-dividing neurons were added to an equal number of proliferating non-neuronal cells, the amount of [methyl-³H]thymidine incorporated by these mixed cultures was 230% greater than that incorporated by 99% pure non-neuronal cultures. Removal of all neurons from such non-neuronal cultures by a 48-h preincubation without nerve growth factor resulted in an even greater increase in [³H]thymidine incorporation upon addition of neurons (370%). When increasing numbers of isolated neurons were added to non-neuronal cell cultures, the amount of [³H]thymidine incorporation initially increased in a dose-dependent fashion until it reached a plateau. In contrast, the addition of increasing numbers of non-neuronal cells to a constant number of neurons resulted in a linear increase in [³H]thymidine incorporation. In some cases neurons and non-neuronal cells were not grown in direct physical contact but were only allowed to communicate with one another through the culture medium. Such indirect communication never resulted in a stimulation of [³H]thymidine incorporation. When neurons were added to cultures of embryonic chick fibroblasts, the neurons grew well but did not stimulate [³H]thymidine incorporation by the fibroblasts. These results suggest that embryonic sympathetic neurons selectively stimulate the proliferation of non-neuronal cells derived from the same source.     

Distribution of [3H]RNA in goldfish optic tectum following intraocular or intracranial injection of [3H]uridine. Evidence of axonal migration of RNA in regenerating optic fibers.

The distribution of [³H]RNA in the goldfish optic tectum following eitherintra-ocular orintracranial injection of [³H]uridine during optic fiber regeneration has been studied by light (LMA) and electron (EMA) microscopic autoradiography.In one group of 4 fish both optic nerves were crushed, and 18 days later [³H]uridine was injected into the right eye. A second group of 5 fish, in which only one optic nerve had been crushed, received intracranial injections of [³H]uridine 18 or 22 days after the crush. All fish were sacrificed 24 days after crushing the optic nerves, a time when regenerating optic fibers have entered the tectum and are establishing functional reconnections. Tecta were fixed in situ with glutaraldehyde, dissected out, and samples were processed for LMA and EMA. Controls were carried out to ensure that [³H]RNA was the only radioactive component present in the tissue after fixation.The distribution of silver grains related to [³H]RNA in intraocularly injected goldfish was different from that following intracranial injection. Following intraocular injection virtually all the [³H]RNA was located in the layers of the left optic tectum (contralateral to the side of intraocular injection) where the regenerating optic fibers course and terminate, whereas virtually no radioactivity was present in the right optic tectum. EMA quantitative analysis of the labeled layers of the left optic tectum revealed that perikarya of cells, most of which are glial cells, had a density of grains related to [³H]RNA of 20–28 g/100 sq.μm; axonal growth cones had a density of 14–24 g/100 sq.μm. Grain densities over non-axonal cell structures were markedly lower, ranging between 3 and 6 g/sq.μm. Grains located over axons and growth cones accounted for 50–60% of all counted grains.Inintracranially injected goldfish, either 2 or 6 days after injection, silver grains were clustered over leptomeninges as well as vessels and parenchymal cells of the tectal strata containing the regenerating optic fibers. In the stratum opticum a high grain density was seen over glial cells, whereas virtually no grains were present over the fascicles of regenerating axons. EMA quantitative analysis revealed a grain density over glial and other parenchymal cells of the stratum opticum of 67 g/100 sq.μm, whereas densities over growth cones and regenerating axons were 1.3 g/100 sq.μm and 1.8 g/100 sq.μm respectively. Grains located over axons and growth cones accounted for 3.3% of all counted grains.On the basis of the present and previous findings it is suggested that followingintraocular injection of [³H]uridine the [³H]RNA present inside regenerating optic axons is transported from the ganglion cells of the retina; on the other hand, the [³H]RNA present in surrounding glial cells is the result of local utilization of [³H]RNA precursors which also migrate from the retina along with the [³H]RNA.It is also concluded that 2 and 6 days followingintracranial injection of [³H]uridine no substantial tranfer of [³H]RNA from glial cells to regenerating optic fibers occurs in the goldfish optic tectum.     

Differential blood-brain barrier permeabilities to [14C]sucrose and [3H]inulin after osmotic opening in the rat

The blood-brain barrier (B-BB) in 3-month-old rats was opened unilaterally by infusing 1.8 ml(+)arabinose in water into the internal carotid artery through a catheter in the external carotid. Two poorly penetrating uncharged test radiotracers of differing molecular weight and size, [¹⁴C]sucrose (340 daltons, radius 5 Å) and [³H]inulin (5500 daltons, radius 15 Å), were simultaneously injected i.v. in untreated rats, or in rats at 1, 30, or 50 min after infusion of hypertonic arabinose solution. Evans-blue solution was injected 5 min prior to osmotic treatment as a visual indicator of barrier integrity. In regions of uninfused control brains, the [¹⁴C]sucrose permeability-surface area (PA) product approximated 10^?5 s^?1, whereas PA was not measurable for [³H]inulin. In arabinose-infused animals, PA products on the ipsilateral hemisphere for both [¹⁴C]sucrose and [³H]inulin were markedly elevated 6 min after infusion, but decreased by 35 and 55 min. In nearly all regions, statistically significant differences were not found between 6-min [¹⁴C]sucrose- and [³H]inulin-PA values (P > 0.05). However, at 35 and 55 min in most regions, the PA for [³H]inulin was significantly lower (P < 0.05) than PA for [¹⁴C]sucrose. The results indicated that the B-BB closed more rapidly to larger than to smaller molecules after osmotic treatment and were consistent with a pore model for osmotic B-BB opening.     

Neurotrophic effects of sciatic nerve extract on denervated extensor digitorum longus muscle in the rat

The trophic action of nerves upon muscles may be mediated by substances secreted by the terminals of the nerves. Several investigators showed that peripheral nerves contain a protein with trophic actions on denervated muscle maintained in tissue culture. This study was undertaken to determine whether or not the neurotrophic properties of proteins extracted from nerves are also demonstrable Pitin vivo. The right extensor digitorum longus (EDL) was denervated in 42 rats. For the following 7 days the denervated EDL was injected daily with a saline extract of rats' sciatic nerves (protein content 2.0 mg/ml). Control groups received injections of saline or of heat-inactivated nerve extract into the denervated EDL, or received no treatment. In denervated EDL muscles injected with nerve extract, there were significantly smaller mean decreases in wet weight (P < 0.05) and total protein (P < 0.05) than in the denervated controls. The mean reduction in cross-sectional area of type IIB muscle fibers (in sections stained for ATPase) was 16 ± 2% less (P < 0.005) than in the control animals. There were no significant differences in the cross-sectional area of IIA fibers or in the content of acetylcholinesterase. Thus, a substance present in a peripheral nerve is able in vivo to ameliorate some of the consequences of denervation of a striated skeletal muscle.     

Quantitative ultrastructural stereology of synapses in nucleus dorsalis after a peripheral nerve injury at birth

Utilizing recent techniques in quantitative stereology, this investigation studied the synaptology of nucleus dorsalis (Clarke's column) in 12-week-old rats whose sciatic nerves were crushed in the 1st postnatal day. Four morphometric variables were analyzed at the levels of L1 and L3 spinal cord segments: total surface area of synaptic contact zones per unit volume (S_V), total length of synaptic contact zones per unit area (L_A), average length of synaptic membrane ($\text{L}$), and numerical density of synapses per unit volume (N_V). The original raw data were corrected for Holmes's effect. The results indicated that peripheral nerve crush at birth induced a transganglionic change in central sensory terminals with a loss of numerous synapses. A significant loss (P < 0.001) of about 32% in the S_V and L_A and a significant loss (P < 0.001) of about 36% in the N_V were observed on the experimental side. There was no preferential loss of synapses in either segment. The mean synaptic membrane length showed no significant difference between the control and experimental sides. The control values of the four morphometric variables calculated for L3 were lower than those calculated for L1. The loss of synapses after a peripheral nerve lesion was probably due to the loss of sensory neurons and their central processes, but there were other possibilities.     

Mitogenic effect of myelinated vs unmyelinated garfish nerve extracts

Mitogenic activities in crude extracts of unmyelinated olfactory nerves and myelinated trigeminal nerves of the garfishLepisosteus osseus were compared. Extracts of each nerve type were added in a range of protein concentrations to serum-starved, subconfluent cultures of BALB/c 3T3 cells. At low protein concentrations (50–250 μg/ml) myelinated nerve extracts produced more [³H]thymidine incorporation in the cultured cells than unmyelinated nerve extracts, while at higher concentrations (500–1000 μg/ml), the latter caused as much DNA synthesis as the myelinated nerve extracts, surpassing them at the highest concentrations tested. The results suggest that both axonal and myelin components contribute to the growth-promoting activity in nerve tissue.     

Regeneration of vomeronasal nerves into the main olfactory bulb in the mouse

Vomeronasal neurosensory cells which are continuously formed in adult mice have been shown to possess axons running in the vomeronasal nerves, since they undergo a reaction of retrograde cell death after vomeronasal nerve transection. Retrograde axonal transport of horseradish peroxidase has been combined with [³H]thymidine labelling of dividing cells to show that the axons of the newly-formed neurosensory cells reach their appropriate target, the accessory olfactory bulb.     

Distribution of serotonin and dopamine receptors in aplysia tissues: Analysis by [3H]LSD binding and adenylate cyclase stimulation

The distribution of receptors for serotonin and dopamine has been studied in various neuronal and non-neuronal tissues from Aplysia californica using: (1) a [³H]LSD binding assay; and (2) stimulation of adenylate cyclase activity. High levels of specific [³H]LSD binding were found in all ganglia and nerves examined. Lower levels of binding were present in a number of muscle tissues and in the sheath surrounding the central ganglia. The ability of serotonin and dopamine to inhibit [³H]LSD binding depended upon the tissue examined. In muscle tissue, most of the binding was sensitive to serotonin. In contrast, a number of ganglia (e.g. the pleural, abdominal or cerebral) contained a considerable proportion of dopamine-sensitive binding. A limited pharmacological analysis of serotonin-sensitive [³H]LSD binding indicated that Aplysia serotonin receptors are closely related to those found in the snail, Helix pomatia, and in rat brain. Adenylate cyclase activity in membranes from Aplysia ganglia, muscles and connective nerves was stimulated by serotonin (but not by dopamine). The amount of serotonin-sensitive adenylate cyclase correlated well with the amount of serotonin-sensitive [³H]LSD binding in most tissues.d-LSD was a partial agonist on the serotonin-sensitive adenylate cyclase, whereas the pharmacologically inactive stereoisomer l-LSD was without effect.The high density of serotonin receptors in pleuro-abdominal connective nerves, and their presence in the connective tissue sheaths surrounding the ganglia, suggests that not all of these receptors are located at synapses. On the other hand, the tissue distribution of dopamine and serotonin receptors, as measured by these techniques, is consistent with that expected from electrophysiological data.     

The amide of the naturally occurring opioid [Met]enkephalin-Arg6-Phe7 is a potent analog of the molluscan neuropeptide FMRFamide

The molluscan neuropeptide FMRFamide (Phe-Met-Arg-Phe-NH₂), the opioid [Met]enkephalin, and two peptides analogous to both were tested on four isolated molluscan muscles. Both FMRFamide and [Met]enkephalin-Arg⁶-Phe⁷-NH₂ (YGG-FMRFamide) excited the ventricle of $\text{Macrocallista nimbosa}$ and inhibited that of $\text{Lampsilis claibornensis}$. These same two peptides also caused contractures of the anterior byssus retractor muscle (ABRM) of $\text{Mytilus edulis}$ and the radula protractor muscle of $\text{Busycon contrarium}$. The effect on the ABRM was a catch contracture, relaxed by 5-hydroxytryptamine. In all cases, the two peptides were equipotent and their actions were identical. Neither [Met]enkephalin, nor the naturally occurring opioid peptide [Met]enkephalin-Arg⁶-Phe⁷ (YGG-FMRF), affected these preparations. In conclusion, these tissues are specifically sensitive to the structure at the C-terminal of FMRFamide, but not to its N-terminal or that of the enkephalins; that is, FMRFamide receptors, but not opioid ones, are present. Nevertheless, the amino acid sequence common to [Met]enkephalin-Arg⁶-Phe⁷ and FMRFamide implies that these two peptides and their naturally occurring congeners are homologous molecules.     

Effect of bradykinin and thrombin on prostacyclin synthesis in endothelial cells from calf and pig aorta and human umbilical cord vein

We have discovered that bradykinin is effective in causing the synthesis of prostacyclin in endothelial cells cultured from calf and pig aorta and human umbilical cord vein. Maximal stimulation is attained at 10 ng bradykinin per ml with a 10 fold increase of PGI₂ as assayed by radioimmunoassay for 6-k-PGF_1α. Ionophore A23187, trypsin, and the precursor arachidonic acid are also stimulatory. We have confirmed the study of Weksler $\text{et al}$ ($\text{J. Clin. Invest., 62}$, 923, 1978) and Czervionke $\text{et al}$ ($\text{Thrombosis Research, 14}$, 781, 1979) that thrombin is effective in stimulating PGI₂ synthesis in endothelial cells from human umbilical cord vein. However, we found that cells from calf or pig aorta are not stimulated as well by thrombin. Thus, there appears to be a difference in the response to thrombin between these cells. When calf cells were grown in the presence of [³H]arachidonic acid, the radioactivity is incorporated mainly into the phospholipids. Bradykinin, ionophore A23187, and trypsin stimulated the release of radioactive materials into medium from [³H]arachidonic acid labeled calf cells. Arachidonic acid is the major radioactive substance released and PGI₂ is the major known arachidonic acid metabolite formed.     

Regulation of Schwann cell proliferation in cultured segments of the adult rat sciatic nerve

Schwann cell proliferation was studied in cultured segments of the rat sciatic nerve by measurement of [³H] thymidine incorporation or through bromodeoxyuridine-(BrdU)-labelling and immunocytochemistry. The aim was to delineate mechanisms involved in the injury-induced proliferative response of Schwann cells. Removal of extracellular Ca²⁺ by addition of EGTA to the culture medium suppressed [³H] thymidine incorporation as did the calmodulin inhibitor 48/80. The Ca²⁺ ionophore A23187 increased incorporation. Staurosporin, an inhibitor of protein kinase C (PKC), suppressed [³H] thymidine incorporation while phorbol-12-myristate-13-acetate (PMA) enhanced incorporation. Manipulation of the cAMP system showed that increased cAMP levels inhibited proliferation. Inhibition of protein kinase A by HA 1004 increased the incorporation of [³H] thymidine. Immunostaining for BrdU and glial specific markers together with morphological evaluation of myelin association showed that proliferation occurred in Schwann cells. The results are consistent with a model in which Schwann cell proliferation is enhanced by Ca²⁺ through activation of calmodulin-dependent and/or PKCdependent mechanisms. Inhibition is achieved through the cAMP system. Together, these results show that Schwann cells regulate proliferation differently in an integrated environment, e.g. the nerve structure, than in isolation as primary monocultures. J. Neurosci. Res. 52:530–537, 1998. © 1998 Wiley-Liss, Inc.     

GABA receptors in bovine cerebral blood vessels: Binding studies with [3H]muscimol

[³H]Muscimol, a potent GABA agonist used to label GABA receptor sites in brain and invertebrate striated muscle, was found to bind specifically to sites in a crude membrane fraction prepared from bovine cerebral blood vessels. Specific [³H]muscimol binding was saturable of high affinity (K_d = 41 nM), and was selectively inhibited by GABA, specific GABA agonists, and the antagonist bicuculline with potencies similar to what has been found for GABA receptors in mammalian brain. GABA and several GABA agonists including muscimol have been reported to dilate isolated cerebral arteries, but not peripheral blood vessels. The pharmacology of the [³H]muscimol binding site correlated well with that of the vasodilatory response. No significant specific [³H]muscimol binding was detected in aorta and mesenteric arteries. The characteristics of the cerebrovascular muscimol binding site thus are indicative of a physiologically relevant GABA receptor associated with cerebral blood vessels. These findings suggest a direct role for GABA in cerebral vascular function.     

3H]Ro5-4864 benzodiazepine binding in the kainate lesioned striatum and Huntington's diseased basal ganglia

[³H]Ro5-4864-labeled peripheral-type benzodiazepine binding sites in the brain were studied after kainic acid lesions of the rat striatum. Following intrastrial kainate injections [³H]Ro5-4864 binding increased to approximately 1000% of control over a period of 1 week and was maintained at this level for up to 6 weeks. Two weeks after lesioning the number of binding sites (B_max) was selectively increased while the dissociation constant (K_d) was only minimally affected. [³H]Ro5-4864 binding in the Huntington's diseased (HD) basal ganglia was not changed as compared to non-neurological control in the caudate nucleus and globus pallidus. A highly significant 51% increase was found in the HD putamen. It is concluded that the peripheral-type, Ro5-4864-sensitive benzodiazepine receptor in the brain may be predominantly localized on glial elements.     

Selective sparing of later-born ganglion cells after neonatal transection of the infraorbital nerve

A combination of [³H]thymidine labelling and retrograde tracing with either horseradish peroxidase (HRP) or true blue (TB) was used to determine whether V primary afferent neurons born on different embryonic (E) days were differentially susceptible to neonatal transection of the infraorbital nerve (ION). In one experiment, rat fetuses were exposed to [³H]thymidine on E-8.5, 9.5, 10.5, 11.5, 12.5, 13.5, 14.5, or 15.5, the left infraorbital nerve (ION) was transected on the day of birth, and both the regenerate and intact IONs were labelled with HRP when the animals reached adulthood. The percentage of HRP labelled cells that were also heavily labelled by [³H]thymidine was calculated for both the intact ganglion and that ipsilateral to the damaged nerve for each animal. A consistently higher percentage of double labelled cells on the lesioned rather than on the intact side for a given E-day was taken as an indication that cells born on the day in question had an increased probability of survival relative to the entire population of V ganglion cells that contributed axons to the ION. Cells born late in gestation on E-12.5 through 14.5 were significantly more likely than early born (E-9.5 through 11.5) cells to survive neonatal axotomy. In a second experiment, fetuses were exposed to [³H]thymidine on either E-9.5, E-10.5, or E-14.5, the vibrissa pads on both sides of the face were injected with TB within 6 hours of birth, and the ION was transected 6–8 hours later. When these rats reached at least 60 days of age, ganglia were processed for the visualization of both TB and [³H]thymidine labelled neurons. Cells labelled with both tracers would have been born on a given E-day, projected to the vibrissa pad via the ION at the time of nerve transection, and survived any naturally occurring or lesion-induced cell death. As in the HRP tracing experiment, ganglion cells born on E-14.5 were significantly more likely to survive neonatal ION transection than those born on either E-9.5 or E-10.5. © 1993 Wiley-Liss, Inc.     

In Vivo release by vagal stimulation of L-[3H] glutamic acid in the nucleus tractus solitarius preloaded with L-[3H] glutamine

In anesthetized and paralyzed rats, using a push-pull perfusion technique, we examined the effect of bilateral vagal stimulation on the release of L-[³H] glutamic acid (L-[³H] Glu) from the nucleus tractus solitarius (NTS), after preloading the tissue either with L-[³H] Glu or L-[³H] glutamine (L-[³H] Gln). Vagal stimulation sufficient to produce a maximum fall of arterial pressure (AP) evoked release of L-[³H] Glu from the NTS when the tissue was preloaded with either ³H-Glu or ³H-Gln, and of D-[³H] aspartic acid (D-[³H) Asp) when this stable Glu analogue was used to preload the tissue. Results demonstrate that the precursor L-Gln is a good marker of the releasable pool of L-Glu in vivo and are consistent with the hypothesis that L-[³H] Glu is a neurotransmitter in the NTS, mediating the vasodepressor response from cardiopulmonary mechanoreceptors.