Postlesion Day Research Articles

Immunocytochemical localization of S-100b protein in degenerating and regenerating rat sciatic nerves.

We studied the cellular and subcellular distribution of S-100b protein in normal, crushed, and transected rat sciatic nerves by an immunocytochemical procedure. In uninjured nerves, S-100b protein was restricted to the cytoplasm and membranes of Schwann cells, with no reaction product present in the nucleus or in axons. Similar images were seen from the first to the thirtieth day after the crush in activated Schwann cells during the degeneration period, i.e., up to the seventh post-lesion day, and in normal Schwann cells reappearing during the regeneration period, i.e., after the seventh post-lesion day, in the zone of the crush and proximal and distal to it. By the technique employed, there seemed to be no differences in the intensity of the immune reaction product in normal and activated Schwann cells. Also, similar images were seen in the proximal stump of transected nerves. Only a slight S-100b protein immune reaction product could be observed in the rare activated Schwann cells present in the distal stump around the seventh post-lesion day, the majority of cell types being represented by fibroblasts and elongated cells at this stage and thereafter. By immunochemical assays, similar results as those presented here have been reported and interpreted as indicative of the presence of S-100 protein in axons or, alternatively, of axonal control over expression of S-100 protein in Schwann cells. Our immunocytochemical data clearly show that the strong reduction in the S-100 protein content of the distal stump of transected nerves is owing to the paucity of Schwann cells and to the decrease in the S-100 protein content of these cells, rather than to degeneration of axons.

Aberrant phosphorylation of neurofilaments accompanies transmitter-related changes in rat septal neurons following transection of the fimbria-fornix

Lesions of the fimbria-fornix (FF) have been reported to cause retrograde changes in neurons of the medial septal nucleus (MSN). To analyze the nature and time course of these events, we investigated changes in cytoskeletal elements (phosphorylated and nonphosphorylated neurofilament (NF) proteins) and transmitter-related enzymes (choline acetyltransferase (ChAT) in MSN neurons following FF transection. During the first week postlesion, ChAT immunoreactivity and size of many perikarya were reduced. Irregular, swollen cholinergic fibers appeared first at postlesion day 2 in caudal septum and soon spread rostrally, reaching rostral septum by day 7. A few perikarya developed abnormal accumulations of phosphorylated NFs. At postlesion days 7–10, many neurons did not stain for ChAT. Phosphorylated NFs were present in many perikarya. At this time, cell loss was apparent in Nissl-stained material. Cholinergic cell loss continued through postlesion week 6–8 but at a much slower rate than during the first week. Phosphorylated NF accumulations in MSN perikarya persisted until postlesion week 6, disappearing thereafter. Double-immunostaining procedures showed that MSN neurons expressed both ChAT and phosphorylated NF immunoreactivity at postlesion day 3; however, at days 7 and 14, cells that accumulated phosphorylated NFs did not stain for ChAT. The results of this study indicate that FF transection leads to perikaryal shrinkage with loss of ChAT immunoreactivity, perikaryal phosphorylation of NFs, cholinergic fiber abnormalities, and cell loss. Recent evidence suggests that reduction of transmitter markers and aberrant phosphorylation of NFs may be involved in the pathogenesis of several neurodegenerative disorders, including Alzheimer's disease. Therefore, FF transection provides a useful animal model for further investigations of complex disorders of the central nervous system that involve degeneration of transmitter-specific pathways.

Trauma‐induced proliferation of astrocytes in the brains of young and aged rats

The time-course and magnitude of astrocyte proliferation following neural trauma was evaluated in young adult (3 months) and mid-aged (16-19 months) male Fischer 344 rats. One to 4 days after a needle wound was made through the cortex and the hippocampus, rats received three intraperitoneal injections of 3H-thymidine at 8 hour intervals and were sacrificed 1 hour after the last injection. For astrocyte quantification, 3H-thymidine autoradiography was combined with immunohistochemical staining for glial fibrillary acidic protein followed by semithin sectioning. In areas of the cortex and hippocampus adjacent to the wound, astrocytes were categorized as unlabeled or labeled with silver grains over the nuclei. Labeling index and numerical density of astrocytes were determined using stereological methods. The results showed that in both young and older rats, astrocyte proliferation is an early glial response to neural trauma, occurring during the first 4 postlesion days and contributing to an increase in astrocyte population. Regional differences in labeling index and numerical density suggest heterogeneity in the proliferative capacity of astrocyte subpopulations in the rat brain. Compared with young animals, older rats demonstrated greater labeling in the cortex but not in the hippocampus. Thus, aging is associated with region-specific increase in astrocyte reactivity to trauma possibly due to increased availability of mitogens or enhanced sensitivity of astrocytes to mitogenic signals.

Neurotoxic lesions of the dorsolateral pontomesencephalic tegmentum-cholinergic cell area in the cat. II. Effects upon sleep-waking states

Kainic acid was injected bilaterally (4.8 μg in 1.2 μl each side) into the dorsolateral pontomesencephalic tegmentum of cats in order to destroy the cholinergic neurons located in that region and thus to study the effects of their destruction upon sleep-waking states. The kainic acid produced a large area of nerve cell loss and/or gliosis centered in the dorsolateral tegmentum-cholinergic cell area, that includes the pedunculopontine tegmental (PPT) and laterodorsal tegmental (LDT) nuclei rostrally (A1-P2), and the parabrachial (PB) and locus coeruleus (LC) nuclei caudally (P3-P5). The mean loss of choline acetyltransferase (ChAT)-immunoreactive neurons within this area was 60% with a range from 25% to 85% across 11 cats. The mean loss of tyrosine hydroxylase (TH)-immunoreactive neurons, differentially distributed through the same region, was 35% with a range of 0–50%. Whereas the kainic acid lesions appeared to have only slight effects upon wakefulness and slow-wave sleep, they had marked effects upon paradoxical sleep (PS), which varied in degree across animals. In cats with the most extensive destruction of cholinergic neurons, PS was eliminated in the first few weeks following the lesion and then reappeared as isolated episodes characterized by sparse, low amplitude PGO spikes in association with few eye movements and an activated cortex, though in absence of neck muscle atonia. Although these PS-like episodes varied in amount, they were significantly less than baseline PS in percent and in duration for the group of 11 animals over one month recording. The PGO spike rate was significantly reduced; the EMG amplitude was significantly increased, marking a loss of neck muscle atonia. The percent of PS-like epochs, the rate of PGO spiking and the EMG amplitude on postlesion day 28 were found to be significantly correlated with the volume of the lesion within the dorsolateral pontine tegmentum-cholinergic cell area. The percent PS-like episodes and PGO spike rate were significantly correlated with the number of remaining ChAT-immunoreactive neurons, but not with the number of remaining TH-immunoreactive neurons within this region. These results suggest that cholinergic pontomesencephalic neurons may be critically involved in the generation of paradoxical sleep and its phasic events.

Effect of bilateral decortication on nerve growth factor content in basal nucleus and neostriatum of adult rat brain

The content of nerve growth factor (NGF) was measured by a sensitive two-site enzyme-linked immunosorbent assay in the basal nucleus and neostriatum at various times following bilateral suction lesions of the cerebral neocortex in adult rats. At 14 and 29 postlesion days, NGF levels in basal nucleus were significantly elevated by 270 and 126%, respectively. In addition, 29 days after cortical lesions, NGF content in the neostriatum was increased 145% above controls. Thus, enhanced NGF production occurs in both basal nucleus and neostriatum of adult rats in response to antero- and/or retrograde neuronal degeneration.

Recovery of escape locomotion following a CNS lesion in Aplysia

The recovery of escape locomotion in Aplysia following a CNS lesion was investigated. The connectives between the cerebral and pleural ganglia were crushed in anesthetized animals, producing a specific behavioral deficit. Animals with lesions failed to initiate escape locomotion in response to tail shock. Tail withdrawal and inking which were also evoked by tail shock were still present. Other behaviors such as normal locomotion and feeding were not impaired. There was gradual recovery from the effects of the lesion. Animals with lesions began to respond to tail shock with weak pedal waves at long latencies after 7-13 days. The responses grew more vigorous and the latencies decreased over subsequent days. Full escape locomotor responses were observed as early as 15 days postlesion. By Postlesion Day 27, all of the animals had completely recovered and gave full escape responses. The mean latency of the escape locomotor response in recovered animals was not significantly different from prelesion control values. In behaviorally recovered animals, retrograde tracing from a point distal to the lesion site stained neurons in the cerebral ganglion. Intracellular dye injections of individual neurons revealed sprouting of new processes. Stimulation of the tail nerve and individual neurons demonstrated synaptic connections between cerebral and pleural ganglia neurons. These results suggest that the observed behavioral recovery was due to pleural ganglia neurons regenerating and forming appropriate synaptic connections in the cerebral ganglion.

Ultracytochemical localization of adenylate cyclase and guanylate cyclase in crushed peripheral nerves.

Cellular and subcellular distribution of adenylate cyclase (AC) and guanylate cyclase (GC) activities in crushed peripheral nerves during regeneration were studied at the electron microscope level. In unlesioned nerves, no AC reaction product could be evidenced, whereas GC was detectable on the plasma membranes of Schwann cells, myelinated and nonmyelinated fibers, and within nonmyelinated axons. At 24 hours after the crush, AC reaction product was found within axonal segments proximal to the zone of the crush in association with mitochondria. At this stage, macrophage-like cells, which probably are transformed Schwann cells, polymorphonuclear leucocytes, and endothelial cells displaying an intense AC reaction product could be detected. On the other hand, at 24 hours after the crush, GC was no longer detectable, except on occasional unlesioned nerve fibers. At 48 hours after the lesion, AC reaction product was no longer detectable within axons, and all AC positivity was associated with plasma membranes of non-neuronal cells, including transformed Schwann cells, occasional macrophages, polymorphonuclear leucocytes, fibroblasts, and elongated cells. As to GC, images similar to those obtained at 24 hours were observed until 48 hours after the crush. From the 7th to the 28th postlesion day, AC activity was localized exclusively to the plasma membranes of fibroblasts and elongated cells. Transformed Schwann cells were no longer detectable, whereas normal Schwann cells and regenerating axons could be seen, and these showed no AC reaction product in analogy to the absence of AC reaction product of unlesioned nerves. During the same period, GC again was detectable on regenerating fibers with the same subcellular localization as that of unlesioned nerves. The present results strongly suggest that starting from the second postcrush day, cells invading the lesioned zone and transformed Schwann cells, all taking part in the formation of the new perineurial tissue, display a high AC activity, which should be taken into account when measuring cyclic adenosine monophosphate (cAMP) levels under these conditions. Also, our data suggest that GC is involved primarily in regeneration processes that occur in crushed peripheral nerves. Thus, the pattern of AC distribution in peripheral unlesioned and lesioned nerves appears to be exactly the opposite of the GC localization examined under similar experimental conditions insofar as nervous fibers are concerned.

Reinnervation of the dentate gyrus and recovery of alternation behavior following entorhinal cortex lesions.

Adult male rats were implanted with chronically indwelling recording electrodes in the dentate hilus of one hemisphere and bipolar stimulating electrodes in the contralateral entorhinal cortex (EC). Daily measurements were then made of the amplitude of responses, evoked through the crossed temporodentate (CTD) pathway, while the rats were unanesthetized and unrestrained. The implanted rats were also trained to alternate turns in a T-maze, with the use of a rewarded-alternation procedure. After reaching criterion performance in the alternation task, each rat was given a lesion of the EC ipsilateral to the recording electrode (n = 14) or a sham lesion (n = 5). Mean amplitudes of the evoked responses increased over Postlesion Days 4-11, probably due to reactive synaptogenesis in the CTD system, reaching a level that was significantly elevated above prelesion levels by Postlesion Day 6. Rats given EC lesions exhibited a transient impairment in alternation performance, with the mean alternation score significantly below prelesion levels on Postlesion Days 2-6. Although 2 EC-lesioned rats did not show a behavioral deficit, the electrophysiological increases and behavioral recovery were correlated in the remaining 12 cases (Pearson r = .73). These results are consistent with the interpretation that sprouting by the CTD system contributes to recovery of T-maze alternation performance following unilateral EC lesions.

The effect of GM 1 ganglioside on cholinergic and serotoninergic systems in the rat hippocampus following partial denervation is dependent on the degree of fiber degeneration

The partial lesion paradigm of the dorsal hippocampal afferents in the rat was used as a model to study the effect of GM 1 ganglioside treatment on recovery of neurotransmitter markers of the cholinergic and serotonirgic activity in various hippocampal regions. It was found that the enhancement of recovery of acetylcholinesterase, choline acetyltransferase and serotonin uptake by GM 1 treatment (30 mg/kg i.m., daily), as studied on the 6th and 21st postlesion day, was dependent on the degree of fiber degeneration. The results may be interpreted in terms of the relationship between the action of GM 1 and that of neuronotropic factors whose release also depends on the extent of the fiber degeneration. These data indicate that GM 1 elicits the recovery of biochemical parameters, or fails to, depending on the specificity of the trauma. The result may explain why, after certain brain lesions, GM 1 does not promote functional recovery.

Effect of forebrain commissurotomy on recovery from unilateral 6-OHDA lesions of the substantia nigra and circling induced by apomorphine

We studied the influence of transection of the interhemispheric fiber systems between the anterior and posterior commissures on recovery from turning behavior induced by a unilateral substantia nigra lesion in rats. It has been argued that the crossed nigrostriatal projection is involved in this recovery, and its site of crossing has been suggested to lie within the region split. Animals which received this transection did not differ from controls with respect to time-course and extent of recovery from lesion-induced asymmetries within the 7 postlesion days examined. Additionally, the commissurotomy did not prevent apomorphine-induced contraversive circling.

Thoracic dorsal funicular lesions affect the bouton patterns on, and diameters of, layer VB pyramidal cell somata in rat hindlimb cortex.

The effect of spinal dorsal funicular lesions (T 12) upon the frequency of boutons on, and diameters of the somata of pyramidal cells in layer VB of hindlimb cortex was studied. Adult rats sustained bilateral damage to either the dorsal column (DC, n = 10) alone or DC combined with the corticospinal tract (CS) (DC + CS, n = 34) and were utilized 1, 2, 3, 7, 14, 30, 45, 60, 90, or 120 days postoperatively (DPO). Neurons randomly sampled from 44 lesioned and 13 unoperated cases were analyzed for the number of silver-impregnated boutons (Rasmussen method) on the circumference of the soma as well as diameters of the soma, nucleus, and nucleolus. Analyses of variance comparing across lesioned and normal groups were significant for bouton counts on the soma (P less than 0.01), and diameters (long axis) of somata (P less than 0.01) and their nuclei (P less than 0.05). Both lesioned groups exhibited significant decreases from normal for these latter three parameters. With respect to survival time for the DC + CS-lesioned animals we noted the following: (1) Bouton counts on the soma significantly decreased below normal between 1 and 60 DPO; this decrease was most dramatic during the first three days postlesion. (2) Somal diameter (long axis) significantly decreased below normal between 2 and 120 DPO (except at 14 and 90 DPO). (3) Nuclear diameter (long axis) significantly decreased below normal only at 90 DPO. (4) Bouton counts on somata of neurons in layers VB and IV [Ganchrow and Bernstein, 1981] of hindlimb cortex correlated negatively and significantly across 120 postlesion days. The rapid shrinkage and reduced afferentation of layer VB somata during the first week following DC + CS lesions suggest initial, retrograde reactions to CS axotomy. Since bouton counts on layer VB somata were significantly less (P less than 0.05) in DC- than DC + CS-lesioned rats, it is hypothesized that CS axotomy regulated a set-point for increased afferentation which was maintained on the shrunken somata between 7 and 120 DPO.

Hypothalamic lesions in the weanling rat alter pancreatic response to glucose

Circulating levels of insulin and glucagon were monitored daily in weanling rats bearing bilateral radiofrequency lesions of the hypothalamic region comprising the ventral pole of the dorsomedial nucleus and at least one third of the dorsal pole of the ventromedial nucleus (V-DMH). Plasma insulin levels in the animals with lesions were significantly elevated by the eighth post-lesion day while plasma glucagon levels were significantly reduced by the 13th day. An intravenous glucose bolus administered to conscious unrestrained animals with lesions had no significant effect on circulating insulin levels but resulted in a dramatic increase in circulating glucagon levels. The IV glucose injections had no significant effect on circulating glucagon levels in the sham-lesioned and unoperated controls while the plasma insulin levels in both control groups were significantly elevated. After a glucose challenge in vitro (300 mg%), insulin release by islets from the lesioned animals showed only a slight increase whereas glucagon release was paradoxically increased. These results provide evidence for an abnormal glucose-sensing function of the pancreatic islet after hypothalamic lesions.

Replication and differentiation of olfactory receptor neurons following axotomy in the adult hamster: a morphometric analysis of postnatal neurogenesis.

Regeneration of olfactory receptor neurons following unilateral olfactory nerve section was studied in Syrian golden hamsters by morphometric procedures. Characteristic structural and histochemical features of olfactory receptor neurons were compared on the sectioned and intact sides of the nasal septum at 6, 12, 18, 33, and 130 days following axotomy. The parameters measured were epithelial thickness and the numbers of nuclei, hematoxylin-staining olfactory vesicles, olfactory marker protein (OMP)-containing neuronal perikarya, and OMP-containing olfactory vesicles. The olfactory receptor neuron population was severely depleted 6 days after axotomy. In the succeeding 12-day period there was an initially rapid, then slower return of receptor neuron numbers. Though the regenerating olfactory epithelium appeared normal by gross inspection 33 days after the lesion, morphometric analysis revealed a substantial increase in the number of olfactory receptor neurons between 33 and 130 days postlesion. At our longest survival interval, all quantitative parameters had returned to 91-99% of control values. The numbers of OMP-containing perikarya and olfactory vesicles on the sectioned side were unchanged between the sixth and 12th postlesion days, thus suggesting that neural turnover was depressed for several days following surgery. Marker protein developed in newly formed receptor neurons between 6 and 12 days after the formation of the olfactory vesicle. Consequently, the ratio between OMP-containing olfactory vesicles and hematoxylin-staining vesicles (O/H) was used as an index of neural differentiation. The O/H ratio on the sectioned side was minimal 12 days following axotomy when stem-cell division was well under way but OMP antigenicity had not yet been expressed in newly formed neurons. At 33 days postlesion, O/H ratios were slightly but significantly greater than the control value, reflecting a degree of synchrony in the regenerating receptor neuron population. The average O/H ratio on the unlesioned side was 0.667. This value, together with our other observations, was used to derive an estimate of the life span of hamster olfactory receptor neurons, approximately 25-35 days.

Strain differences in recovery from sensorimotor neglect after unilateral globus pallidus lesions in mice

Recovery of somatosensory orientation after unilateral electrolytic lesions of globus pallidus was studied in two inbred strains of mice (C57BL/6, Sec 1/ReJ). During the first four post-lesion days mice of both strains did not react to tactile stimuli (von Frey hair of 2 g intensity) applied to the thorax area contralateral to the lesion. Successively, there was a gradual replacement by non-oriented activation and incomplete oriented responses, followed by normal precise orientation. Recovery of orientation proceeded at a significantly higher rate in C57 mice. After a period of four months C57 mice reached a recovery level of 91% in comparison to 56% of Sec mice. The genotype dependent differences of recovery mechanisms are discussed in terms of plasticity and dopaminergic systems.

Experimental percutaneous approach to the trigeminal ganglion in dogs with histopathological evaluation of radiofrequency lesions.

A percutaneous approach via the foramen ovale to the trigeminal ganglion of dog was developed and used to produce radiofrequency lesions analogous to those made for the treatment of trigeminal neuralgia. Histological studies over 14 post-lesion days indicated that neural destruction is more extensive than previously concluded.

Terminal proliferation in the partially deafferented dentate gyrus: time courses for the appearance and removal of degeneration and the replacement of lost terminals.

The time courses for the appearance and removal of degenerating terminals and the loss and reappearance of intact terminals were investigated in the partially denervated inner molecular layer of the dentate gyrus of the adult rat. Dense degeneration was evident in the neuropil within 26 hours following contralateral hippocampectomy. These profiles increased rapidly in number until the maximal degree was reached at two to three days postlesion, after which the degenerating terminals were quickly removed from the neuropil. A more rapid rate of removal occurred during the 3-to 5-day survival period than from 6 to 50 days postlesion. The intact terminal population dropped 35% within two days of the lesion and remained at this level until six to eight days postlesion when the number began to steadily increase. The time course for this reappearance can be divided into two phases: a period of rapid terminal addition from 6 to 15 days followed by a phase of slower acquisition. This recovery continued until the normal synaptic density was regained by 50 to 65 days postlesion. These results indicate that a substantial proportion of degenerating endings are removed well in advance of the time at which terminal proliferation begins, suggesting that certain changes other than merely the removal of competitive inputs must take place prior to growth of new terminals. Possible explanation suggested by the present results for the delay in the onset of sprouting include: (1) an absence of appropriate postsynaptic targets during the 2-to 5-day postlesion period and (2) inhibition of axonal growth by the glial cells which are phagocytizing the degenerating products. Beyond the sixth postlesion day the rate at which new terminals appear does correlate with the rate at which degeneration is removed. This suggests that once underway the time course for sprouting may be determined by the avaiabliity of postsynaptic sites.

Effects of lesions in the pontine tegmentum on the sleep stages in the rat

1. Limited coagulations of the locus coeruleus and subcoeruleus nuclei have been performed in rats and the sleep-waking cycle was continuously monitored during 9 days. The cortical and diencephalic noradrenaline content was mesured at the termination of the experiment, on the 10th post lesion day. 2. The bilateral destruction of the locus coeruleus is followed by the appearance of a uro-genital syndrome consisting of hema turia, bladder distension and penile erection. The states of sleep are disturbed during the first two days (increase of slow-wave sleep and decrease of paradoxical sleep times) and thereafter return to normal. Additionally, an hyperthermia appears during the third experimental day. The cortical and diencephalic noradrenaline content decrease to 70%. 3. The simultaneous lesion of both locus coeruleus and subcoeruleus nuclei is followed by the appearance of an aphagia adipsia syndrome in addition to the uro-genital syndrome. After these lesions, it is no long possible to find paradoxical sleep episodes in polygraphic recordings while the amount of slow wave sleep is normal. Cortical and diencephalic noradrenaline content decrease more than 50%. 4. In normal rats direct injection of 6 hydroxydopamine directly in both locus coeruleus and nuclei subcoeruleus had no discernable effects either on the behaviour or on the states of sleep. The cortical noradrenaline content decreased 30% below control values. 5. These results are consistent with but do not prove the hypothesis that part of the pontine tegmentum might play an important role in triggering paradoxical sleep episodes. The role of these regions in the regulation of internal temperature, food consumption and bladder motricity is also discussed.

Differential behavioral and neurochemical effects following lesions of the dorsal or median raphe nuclei in rats

Locomotor activity and regional forebrain levels of serotonin (5-HT) were measured following the placement of electrolytic lesions in either the dorsal or median raphe nucleus of adult male rats. In the first two experiments, control lesions were placed in the brachium conjunctivum, and in the third experiment, a sham lesion group served as control. Median lesions significantly increased locomotor actiivity as measured in tilt cages, by 250–300% on the second day post-lesion, and this elevation stabilized at approximately 100% above control levels on day 9 post-lesion. There were no statistically significant differences in the amount of locomotor activity in the dorsal, brachium or sham lesioned groups on any post-lesion day. When the amount of 5-HT depletion was measured 5 days post-lesion, it was found that the dorsal (D) and median (M) lesions produced similar reductions in cerebral cortex (D — 40%; M — 31%); hypothalamus (D — 54%; M — 58%) and striatum (D — 50%; M — 29%). However, the effects of the two lesions were markedly different in the hippocampus. The dorsal lesion produced a non-significant 10% reduction in hippocampal 5-HT level, while the median lesion caused an 82% reduction. On the basis of these data it is hypothesized that a reduction in hippocampal 5-HT may account for the increased activity in the median lesioned group.