Comparison of the Protection against Neuronal Injury by Hypothalamic Peptides and by Dexamethasone

The comparative study has been carried out on hypothalamic neurohormone (proline-rich polypeptides-PRP) and synthetic glucocorticoid dexamethasone (DEX) protective properties at the systemic (i/m) administration. Both background and evoked electrical activity (on n.ischiadicus stimulation) of single neurons in the lumbo-sacral part (laminae II–VI and VII–VIII by Rexed) and field potentials (FP) of spinal cord were recorded during acute experiments on intact spinal rats, subjected to Vipera Raddei (VR) venom intoxication, and chronic spinal cord trauma (hemisection). The action of PRP was characterized by the pronounced activation of the background activity (BA) with adaptive effect, depending on dose and initial level of BA, by results of the statistical analysis. A high effect is received from comparatively small doses. For comparison it was used strong glucocorticoid DEX, possessing single-directed but less expressed excitative action on investigated spinal cord neurons. The initial increase of BA frequency with subsequent depression was the typical symptom of venom influence. A protective effect of preliminary PRP injection is revealed on the succeeding VR venom influence. Use of PRP and DEX causes the increase of reduced activity of neurons on the injury side of animals with spinal cord hemisection. It provides the possibility of the therapeutic utilization. It was revealed considerably more expressed PRP action on neurodegenerative process connected to spinal cord injury (in comparison with DEX). The influence of hormones was compared in identical conditions of experiments on non-injured (control) and injured sides. Taking into consideration revealed protection characteristic of PRP and also the ability of snake venom to stabilize and to prolong its action combined with these preparations, the assumption is made on prospective use of the specified combination in clinical practice.     

Protection Against Snake Venom-Induced Neuronal Injury by the New Hypothalamic Neurohormone

The action of PRP is characterized by the pronounced activation of the background activity (BA) of the brain spinal cord, and the degree of the activity depends on BA initial level. The typical peculiarity of Vipera raddei venom influence is the initial increase in frequency of BA with subsequent depression. A preliminary injection of PRP has a protective effect at subsequent influence of venom. In animals with hemisection the PRP increases the decreased activity of neurons on injury side. Taking into consideration the protective peculiarities of PRP in the relationship to snake venom and the possibility of the latter to stabilize and prolong the action of drugs (in the case of PRP) combined with them, it is supposed that the mentioned use of the combination in clinical practice will be perspective. The data obtained testify the PRP to be a neuroprotector against many toxic compounds formed in organism (glutamate, ceramid, beta-amyloid neurotoxisity, etc.). Investigations in this aspect are still in the process.     

The Effect of a Spinal Cord Hemisection on Changes in Nitric Oxide Synthase Pools in the Site of Injury and in Regions Located Far Away from the Injured Site

1. The present study was designed to examine the nitric oxide synthase activities (constitutive and inducible) in the site of injury in response to Th10-Th11 spinal cord hemisection and, to determine whether unilateral disconnection of the spinal cord influences the NOS pools on the contra- and ipsilateral sides in segments located far away from the epicentre of injury.2. A radioassay detection was used to determine Ca²⁺-dependent and inducible nitric oxide synthase activities. Somal, axonal and neuropil neuronal nitric oxide synthase was assessed by immunocytochemical study. A quantitative assessment of neuronal nitric oxide synthase immunoreactivity was made by an image analyser. The level of neuronal nitric oxide synthase protein was measured by the Western blot analysis.3. Our data show the increase of inducible nitric oxide synthase activity and a decrease of Ca²⁺-dependent nitric oxide synthase activity in the injured site analysed 1 and 7 days after surgery. In segments remote from the epicentre of injury the inducible nitric oxide synthase activity was increased at both time points. Ca²⁺-dependent nitric oxide synthase activity had decreased in L5-S1 segments in a group of animals surviving for 7 days. A hemisection performed at thoracic level did not cause significant difference in the nitric oxide synthase activities and in the level of neuronal nitric oxide synthase protein between the contra- and ipsilateral sides in C6-Th1 and L5-S1 segments taken as a whole. Significant differences were observed, but only when the spinal cord was analysed segment by segment, and/or was divided into dorsal and ventral parts. The cell counts in the cervicothoracic (C7-Th1) and lumbosacral (L5-S1) enlargements revealed changes in neuronal nitric oxide synthase immunoreactivity on the ipsilateral side of the injury. The densitometric area measurements confirmed the reduction of somal, neuropil and axonal neuronal nitric oxide synthase immunoreactive staining in the ventral part of rostrally oriented segments.4. Our findings provide evidence that the changes in nitric oxide synthase pools are limited not only to impact zone, but spread outside the original lesion. The regional distribution of nitric oxide synthase activity and neuronal nitric oxide synthase immunoreactivity, measured segment by segment shows that nitric oxide may play a significant role in the stepping cycle in the quadrupeds.     

Neuroprotective Action of Hypothalamic Peptide PRP-1 at Various Time Survivals Following Spinal Cord Hemisection

The purpose of the present study was to evaluate the neuroprotective action of proline-rich peptide-1 (PRP-1) produced by hypothalamic nuclei cells (nuclei paraventricularis and supraopticus) following lateral hemisection of spinal cord (SC). The dynamics of rehabilitative shifts were investigated at various periods of postoperative survival (1–2, 3, and 4 weeks), both with administration of PRP-1 and without it (control). We registered evoked spike flow activity in both interneurons and motoneurons of the same segment of transected and symmetric intact sides of SC and below it on the stimulation of mixed (n. ischiadicus), flexor (n. gastrocnemius) and extensor (n. peroneus communis) nerves. In the control group (administration of 0.9% saline as placebo), no significant decrease of post-stimulus activity of neurons was observed on the transected side by the 2nd week. This activity strongly decreased by week 3 postaxotomy, with some increase on the intact side, possibly of compensatory origin. No shifts occurred by the 4th week. Regardless of the period of administration, PRP-1 increased neuronal activity on the transected side, with the same activation levels on both SC sides. These data were confirmed by histochemical investigation. PRP-1 administration, both daily and every other day, for a period of 2–3 weeks led to prevention of scar formation and promotion of the re-growth of white matter nerve fibers in the damaged area. It also resulted in prevention of neuroglial elements degeneration and reduction in gliosis expression in the lesion supporting neuronal survival. Thus, PRP-1 achieved protection against “tissue stress”, which was also confirmed by the registration of activity on the level of transection and restoration of the motor activity on the injured side. The obtained data propose the possibility of PRP-1 application in clinical practice for prevention of neurodegeneration of traumatic origin.     

灵芝孢子和L-NNA对脊髓损伤后背核线粒体细胞色素氧化酶活性的影响

目的探讨灵芝孢子和一氧化氮合酶 (NOS)抑制剂L-NNA联合应用对大鼠脊髓半横断后受损伤的背核线粒体细胞色素氧化酶活性的影响.方法将20只SD成年雌性大鼠(200-250g)行右侧T11脊髓半横断30d后,对受损伤脊髓做细胞色素氧化酶酶组化染色;用图像分析方法检测L1脊髓段背核线粒体细胞色素氧化酶活性的变化,用酶组化电镜技术观察L1脊髓段背核细胞色素氧化酶活性的分布位置.结果与对照组相比,L-NNA组和灵芝孢子组L1脊髓损伤侧背核线粒体细胞色素氧化酶活性有所提高,灵芝孢子 L-NNA组损伤侧背核线粒体细胞色素氧化酶活性最大.各组L1脊髓背核细胞色素氧化酶活性均出现在线粒体内,具有细胞色素氧化酶活性的线粒体存在所有神经元胞体及其树突和轴突内,也存在于神经胶质细胞胞体及其突起内.结论灵芝孢子和L-NNA均可提高大鼠脊髓半横断后受损伤的脊髓背核线粒体细胞色素氧化酶的活性,两者联合应用更能提高受损伤的背核线粒体细胞色素氧化酶的活性.     

Changes in expression of ciliary neurotrophic factor (CNTF) and CNTF-receptor α after spinal cord injury

To determine if ciliary neurotrophic factor (CNTF) is involved in the response to spinal cord injury, we studied changes in the expression of CNTF and that of its receptor, CNTF-receptor α (CNTFRα), in the rat spinal cord after a unilateral spinal cord hemisection. Using in situ hybridization, we found that CNTFRα mRNA levels in spinal cord motoneurons increased dramatically by 1 day after hemisecting the spinal cord at T2. This increase in expression was present only in motoneurons caudal, but not rostral, to the lesion. In addition, we detected increased levels of CNTF mRNA in the spinal cord white matter, also by 1 day following injury. Unlike CNTFRα, however, the increase in CNTF mRNA was evident both rostral and caudal to the lesion. Levels of both CNTF and CNTFRα mRNA declined between 1 and 5 days, and by 10 days they were not significantly different from normal animals. These findings suggest that CNTF may play a local role in the response to spinal cord injury. © 1997 John Wiley & Sons, Inc. J Neurobiol 32: 251–261, 1997.     

A damage model for the percutaneous triple hemisection technique for tendo-achilles lengthening

A full understanding of the mechanisms of action in the percutaneous triple hemisection technique for tendo-achilles lengthening has yet to be acquired and therefore, an accurate prediction of the amount of lengthening that occurs is difficult to make. The purpose of this research was to develop a phenomenological damage model that utilizes both matrix and fiber damage and replicates the observed behavior of the tendon tissue during the lengthening process. Matrix damage was triggered and evolved relative to shear strain and the fiber damage was triggered and evolved relative to fiber stretch. Three examples are given to show the effectiveness of the model. Implementation of the damage model provides a tool for studying this common procedure, and may allow for numerical investigation of alternative surgical approaches that could reduce the incidence rates of severe over-lengthening.     

Analysis of Gene Expression Following Sciatic Nerve Crush and Spinal Cord Hemisection in the Mouse by Microarray Expression Profiling

1. The responses of periphery (PNS) and central nervous systems (CNS) towards nerve injury are different: while injured mammalian periphery nerons can successfully undergo regeneration, axons in the central nervous system are usually not able to regenerate.2. In the present study, the genes which were differentially expressed in the PNS and CNS following nerve injury were identified and compared by microarray profiling techniques.3. Sciatic nerve crush and hemisection of the spinal cord of adult mice were used as the models for nerve injury in PNS and CNS respectively.4. It was found that of all the genes examined, 14% (80/588) showed changes in expression following either PNS or CNS injury, and only 3% (18/588) showed changes in both types of injuries.5. Among all the differentially expressed genes, only 8% (6/80) exhibited similar changes in gene expression (either up- or down-regulation) following injury in both PNS and CNS nerve injuries.6. Our results indicated that microarray expression profiling is an efficient and useful method to identify genes that are involved in the regeneration process following nerve injuries, and several genes which are differentially expressed in the PNS and/or CNS following nerve injuries were identified in the present study.     

Assessing Forelimb Function after Unilateral Cervical SCI using Novel Tasks: Limb Step-alternation,Postural Instability and Pasta Handling

Cervical spinal cord injury (cSCI) can cause devastating neurological deficits, including impairment or loss of upper limb and hand function. A majority of the spinal cord injuries in humans occur at the cervical levels. Therefore, developing cervical injury models and developing relevant and sensitive behavioral tests is of great importance. Here we describe the use of a newly developed forelimb step-alternation test after cervical spinal cord injury in rats. In addition, we describe two behavioral tests that have not been used after spinal cord injury: a postural instability test (PIT), and a pasta-handling test. All three behavioral tests are highly sensitive to injury and are easy to use. Therefore, we feel that these behavioral tests can be instrumental in investigating therapeutic strategies after cSCI.     

Immunohistochemical Study of Immunophilin 1–15 Fragment in Intact Frog Brain,and in the Brain and Spinal Cord of Intact and Spinal Cord Hemisectioned Rats

Previously by immunohistochemical technique the distribution of immunophilin 1–15 fragment (IphF) isolated from bovine hypothalamus was examined in various tissues (heart, lung), including immune system organs (spleen and thymus) of intact rats. IphF-like immunoreactivity (IphF-LI) was revealed in several cell types: lymphocytes, monocytes, macrophages and mast cells. In the present study the immunohistochemical localization of IphF was examined in intact rat and frog brains. In rat brain several cell groups concentrated particularly in the supraoptic nucleus (SON) of hypothalamus, medulla oblongata (reticular formation, olives, hypoglossal and facial motor nuclei) and cerebellum (lateral cerebellar nucleus) demonstrated IphF-LI. In frog hypothalamus (SON) the same working dilution (1:5000) of IphF-antiserum revealed very strong immunoreactivity. In the paraventricular nucleus (PVN) IphF-LI varicosities were scattered around the immunonegative cells. The second cell groups showing IphF-LI in the frog brain were gliocytes (mainly the astrocytes). Besides, IphF distribution was investigated in rats subjected to hemisection of spinal cord (SC) with and without administration of proline-rich polypeptide (PRP). PRP was isolated from bovine neurohypophysis neurosecretory granules, produced by magnocellular nuclei of hypothalamus. Hemisection of SC led to changes of IphF distribution in the hypothalamus. In PRP treated animals IphF showed no immunoreactivity. PRP is suggested to act as a neurotransmitter and neuroregulator.