Bilateral bulbospinal projections to pudendal motoneuron circuitry after chronic spinal cord hemisection injury as revealed by transsynaptic tracing with pseudorabies virus

Complications of spinal cord injury in males include losing brainstem control of pudendal nerve-innervated perineal muscles involved in erection and ejaculation. We previously described, in adult male rats, a bulbospinal pathway originating in a discrete area within the medullary gigantocellularis (GiA/Gi), and lateral paragigantocellularis (LPGi) nuclei, which when electrically microstimulated unilaterally, produces a bilateral inhibition of pudendal motoneuron reflex circuitry after crossing to the contralateral spinal cord below T8. Microstimulation following a long-term lateral hemisection, however, revealed reflex inhibition from both sides of the medulla, suggesting the development or unmasking of an injury-induced bulbospinal pathway crossing the midline cranial to the spinal lesion. In the present study, we investigated this pathway anatomically using the transsynaptic neuronal tracer pseudorabies virus (PRV) injected unilaterally into the bulbospongiosus muscle in uninjured controls, and ipsilateral to a chronic (1-2 months) unilateral lesion of the lateral funiculus. At 4.75 days post-injection, PRV-labeled cells were found bilaterally in the GiA/Gi/LPGi with equal side-to-side labeling in uninjured controls, and with significantly greater labeling contralateral to the lesion/injection in lesioned animals. The finding of PRV-labeled neurons on both sides of the medulla after removing the mid-thoracic spinal pathway on one side provides anatomical evidence for the bilaterality in both the brainstem origin and the lumbosacral pudendal circuit termination of the spared lateral funicular bulbospinal pathway. This also suggests that this bilaterality may contribute to the quick functional recovery of bladder and sexual functions observed in animals and humans with lateral hemisection injury.     

X线照射促进损伤脊髓组织修复的实验研究

目的 探讨X线照射对大鼠脊髓损伤(SCI)区组织结构及神经功能恢复的作用.方法 采用Allen打击法建立大鼠SCI模型,46只雌性Spragne-Dawley大鼠均接受T<,11-12>节段SCI.所有人组实验动物随机分为空白组、10 Gy照射组、20 Gy照射组,照射组于SCI后2周接受X线照射,空白组不予照射.各组实验动物分别于SCI后6周、14周处死半数,期间进行运动学、电生理学检测,大鼠处死后取出损伤区脊髓组织进行组织学检查,并对结果进行统计学分析.结果 SCI后6周、14周时照射组(10 Gy、20 Gy)神经丝(NF)计数均较空白组显著增多(P<0.05).各组髓鞘碱性蛋白(MBP)计数14周时较6周显著数少(P<0.05),14周时照射组(10 Gy、20 Gy)较空白组显著减少(P<0.05).各组胶原纤维酸性蛋白(GFAP)、Nngo-A计数14周时较6周时显著增多,照射组与空白组计数差异无统计学意义.运动学、SEP检测结果各组间差异无统计学意义.结论 大鼠SCI后脊髓中枢神经组织结构及功能具有自我修复机制,该进程至少持续14周,X线照射对其具有一定的促进作用.     

Expression and role of PAK6 after spinal cord injury in adult rat

Objective: To observe p21-activated kinase 6 (PAK6) expression and its possible role after spinal cord injury (SCI) in adult rat.Methods: Sprague-Dawley rats were subjected to spinal cord injury. To explore the pathological and physiological significance of PAK6, the expression patterns and distribution of PAK6 were observed by Western blot, immunohistochemistry and immunofluorescence.Results: Western blot analysis showed PAK6 protein level was significantly up-regulated on day 2 and day 4,then reduced and had no up-regulation till day 14. Immunohistochemistry analysis showed that the expression of PAK6 was significantly increased on day 4 compared with the control group. Besides, double immunofluorescence staining showed PAK6 was primarily expressed in the neurons and astrocytes in the control group. While after injury, the expression of PAK6 was increased significantly in the astrocytes and neurons, and the astrocytes were largely proliferated. We also examined the expression of proliferating cell nuclear antigen (PCNA) and found its change was correlated with the expression of PAK6. Importantly, double immunofluorescence staining revealed that cell proliferation evaluated by PCNA appeared in many PAK6-expressing cells on day 4 after injury.Conclusion: The up-regulation of PAK6 in the injured spinal cord may be associated with glial proliferation.     

新型大鼠脊髓打击器的制备及建模大鼠的功能与病理评价

[目的]制备一种简便、实用的急性大鼠脊髓损伤建模装置。[方法]用木板、新泡沫板、吸管、手术缝线及金属插销杆等制作新型大鼠脊髓打击器。取36只成年雌性SD大鼠,体质量为220~250 g,随机等分为假手术组(Sham组)、轻度打击组(SCI1组)和重度打击组(SCI2组),利用自制大鼠脊髓打击器建立大鼠脊髓损伤模型,SCI1组采用10 g×25 mm打击规格进行垂直打击损伤,SCI2组采用10 g×50 mm打击规格进行垂直打击损伤。各组大鼠于术前1 d,术后1、3、7、14、21和28 d进行BBB运动学评分,观察大鼠后肢运动功能情况,术后28 d对各组大鼠受损脊髓组织进行HE染色、神经元核抗原(Neu N)免疫组化检测。[结果]Sham组各观察时间点的BBB评分均为21分,各SCI组术后1 d时BBB评分为0分,之后BBB评分结果随时间延长呈逐步恢复趋势,从3 d开始,SCI1组的BBB评分高于SCI2组(P<0.05)。各组大鼠后肢左右侧活动情况无明显差异,实验过程中无大鼠死亡。术后28 d HE染色、Neu N免疫组化结果显示SCI2组脊髓受损程度明显重于SCI1组(P<0.05)。[结论]该新型大鼠脊髓打击器制作简便、可行性高,建立的SCI模型具有稳定性高、可重复性强、损伤程度可调节等特点,适合相关科研人员使用。     

脊神经前根吻合重建脊髓损伤大鼠神经反射通路的形态学研究

目的:观察利用脊神经前根吻合重建脊髓损伤大鼠股四头肌神经反射通路的形态学情况。方法:取4周龄SD大鼠20只,体重120～150g;将左侧L1神经前根与支配股四头肌的优势脊神经(L3)前根通过尾神经桥接吻合,右侧不作任何处理。神经吻合术后6个月,手术分离神经吻合段。将能分离神经吻合段的大鼠在L2脊髓水平左半侧切断脊髓制备大鼠脊髓半切损伤模型,饲养4周后左侧股神经注射Trueblue(TB)染色剂进行逆行荧光染色,观察L1脊髓前角是否有TB标记的阳性细胞;同时解剖分离神经吻合段进行HE染色及电镜观察。结果:饲养过程中大鼠死亡4只。成功分离出10只大鼠的神经吻合段。吻合段神经肉眼观察光滑圆润,无萎缩;HE染色光镜下吻合段神经纤维排列整齐,走行一致,为典型的有髓神经纤维结构;电镜下吻合段神经纤维具有明显神经轴突特征。TB荧光逆行示踪左侧L1脊髓前角可见荧光标记阳性的神经元,而右侧无标记阳性细胞。结论:L1与L3脊神经前根吻合可成功建立L2脊髓半切损伤大鼠的股四头肌脊髓旁神经反射通路。     

Evaluation of olfactory ensheathing and schwann cells after implantation into a dorsal injury of adult rat spinal cord

Olfactory ensheathing cells (OECs) and Schwann cells (SCs) obtained from adult transgenic rats expressing alkaline phosphatase (AP) were studied following implantation into intact spinal cord and after dorsal column crush (DCC) injury, either within the lesion or near the lesion borders. We observed no evidence of migration of AP OECs or AP SCs after lesion site injections, with most cells remaining in or nearby the injection/lesion site. Acute injection of either cell type outside of the lesion site resulted in the presence of cells in the lesion even two hours after injection. However, after a 2-week delay between DCC injury and cell injection, only OECs injected 2.5-mm outside of a DCC lesion entered the lesion, while SCs did not pass a region of increased astroglial immunoreactivity. GFAP-immunoreactivity also revealed differences in the astroglial scar at the lesion border with openings apparent in this region only in the OEC group. SCs induced greater ingrowth of CGRP-positive axons within the lesion, two weeks post-injury. Equivalent numbers of GAP-43-positive axons grew within the lesion after SC or OEC implantation. These findings show that, although there is no active migration for either cell type, both OECs and SCs are able to support axonal regrowth and/or sprouting into the lesion. The openings in the astroglial boundary at the lesion site may give OECs a potential advantage over SCs in promoting axonal growth through the astroglial scar.     

Effects of enriched housing on functional recovery after spinal cord contusive injury in the adult rat

To date, most research performed in the area of spinal cord injury focuses on treatments designed to either prevent spreading lesion (secondary injury) or to enhance outgrowth of long descending and ascending fiber tracts around or through the lesion. In the last decade, however, several authors have shown that it is possible to enhance locomotor function after spinal cord injury in both animals and patients using specific training paradigms. As a first step towards combining such training paradigms with pharmacotherapy, we evaluated recovery of function in adult rats sustaining a spinal cord contusion injury (MASCIS device, 12.5 mm at T8), either housed in an enriched environment or in standard cages (n = 15 in both groups). The animals in the enriched environment were stimulated to increase their locomotor activity by placing water and food on opposite sides of the cage. As extra stimuli, a running wheel and several other objects were added to the cage. We show that exposure to the enriched environment improves gross and fine locomotor recovery as measured by the Basso, Beattie, and Bresnahan (BBB) locomotor rating scale, the BBB subscale, the Gridwalk, and the Thoracolumbar height test. However, no group differences were found on our electrophysiological parameters nor on the amount of spared white matter. These data justify further studies on enriched housing and more controlled exercise training, with their use as potential additive to pharmacological intervention.     

Central nervous system neurons labeled following the injection of pseudorabies virus into the rat prostate gland

BACKGROUND: The human prostate gland plays an important role in male fertility and is involved in different functional pathologies of the male lower urinary tract (LUT). The role of the prostate in these medical disorders is mainly unknown. Traditional surgical therapeutic attempts often fail to help these patients. For years, the clinical sciences have been stagnating due to a lack of basic science knowledge. Investigations into neuroanatomy and neurophysiology are urgently needed. Therefore, the neuroanatomy of the prostate gland in an experimental setup was explored. Recent progress in neuroscience methodology allows a transneuronal tracing by using a self-amplifying virus tracer, pseudorabies virus (PRV). METHODS: Sixty-two individual adult male Sprague-Dawley rats were used for retrograde transneuronal mapping of the spinal cord and brain stem after PRV-injection and control experiments. A PRV-tracer (5 microl, 1 x 10(8) pfu/ml) was injected into the prostate gland. After a survival time of 72, 96, or 120 hr, the animals were sacrificed. Brain and spinal cord were harvested via a dorsal laminectomy. After cutting on a freezing microtome, the tissue was immunostained for PRV. RESULTS: PRV-positive cells were found within the sacral (S1-S2) and the thoracolumbar (T13-L2) spinal cord. At the supraspinal level, positive cells were found within the following regions: nucleus raphe, lateral reticular formation, nucleus gigantocellularis, A5 noradrenergic cell region, locus coeruleus, pontine micturition center, hypothalamus, medial preoptic region, and periaquaductal gray. CONCLUSIONS: This is the first investigation on the central innervation of the prostate gland showing a broad central representation of neurons involved in the control of the prostate gland. It is obvious, comparing data from the literature, that there is a broad overlap in the innervation of pelvic visceral organs (bladder, rectum, and urethra). The appreciation of these neuroanatomical circumstances allows a growing understanding of common urological pathologies within the pelvis (pelvic pain, lower urinary tract, and bowel dysfunction).     

Depression and spinal cord injury: a monozygotic twin study

Although, in earlier work, depression in individuals with spinal cord injury (SCI) was attributed to difficulties adjusting to SCI, more recent articles have emphasized the importance of constitutional and environmental factors not specific to SCI, as well as established theoretical models of depression. To further explore this question, 11 pairs of monozygotic twins, where one of each pair was spinal cord injured, were studied. Measures included the Beck Depression Inventory, the depression scale of the SCL-90R, and the Rosenberg Self-Esteem Scale. Using pairwise t tests, the authors did not find any significant differences between SCI and non-SCI co-twins. These findings are consistent with the idea that the occurrence of SCI does not inevitably lead to increased depression.     

Mechanical characterization of the injured spinal cord after lateral spinal hemisection injury in the rat

The glial scar formed at the site of traumatic spinal cord injury (SCI) has been classically hypothesized to be a potent physical and biochemical barrier to nerve regeneration. One longstanding hypothesis is that the scar acts as a physical barrier due to its increased stiffness in comparison to uninjured spinal cord tissue. However, the information regarding the mechanical properties of the glial scar in the current literature is mostly anecdotal and not well quantified. We monitored the mechanical relaxation behavior of injured rat spinal cord tissue at the site of mid-thoracic spinal hemisection 2 weeks and 8 weeks post-injury using a microindentation test method. Elastic moduli were calculated and a modified standard linear model (mSLM) was fit to the data to estimate the relaxation time constant and viscosity. The SLM was modified to account for a spectrum of relaxation times, a phenomenon common to biological tissues, by incorporating a stretched exponential term. Injured tissue exhibited significantly lower stiffness and elastic modulus in comparison to uninjured control tissue, and the results from the model parameters indicated that the relaxation time constant and viscosity of injured tissue were significantly higher than controls. This study presents direct micromechanical measurements of injured spinal cord tissue post-injury. The results of this study show that the injured spinal tissue displays complex viscoelastic behavior, likely indicating changes in tissue permeability and diffusivity.     

Reconstruction of the spinal cord and its motor connections using embryonal nervous tissue transplantation and peripheral nerve autotransplantation. A study in the adult rat]

Our research group is studying, in the adult rat, the conditions of an anatomical and functional reconstruction of the spinal cord and of its motor connections, following a spinal lesion that is either small (focal) or large (depletive). In this attempt to repair the damaged neuronal circuitry, we use, alone on in combination, two transplantation techniques, namely that of embryonic neural tissue, to replace the lost neurons, and that of long segments of autologous peripheral nerves to stimulate and guide either the axonal regrowth from injured host spinal neurons or the axogenesis of transplanted embryonic neurons. The common denominator to the whole experimentation is the setting up of a "nerve bridge" (peroneal nerve autograft) joining the injured cervical spinal cord an aneural region of a nearby denervated skeletal muscle. In a first experimental model (focal lesion), in which only a peripheral nerve autograft is used, it can be observed that local injured (or uninjured?) motoneurons have the actual capacity to extend axons throughout the nerve bridge and, thus, to reach the muscle and reform functional and stable, mainly ectopic, neuromuscular connections. In a second experimental model (depletion lesion) a cavity is made, by suction, in the cervical spinal cord, thus causing a damage which resembles, in some respects, certain types of neurodegenerative spinal lesions. This cavity is filled with different kinds of embryonic neural transplants. The surviving transplanted neurons differentiate axonal projections, some of them extending into the peripheral nerve bridge. Studies aimed at determining the capacities of motor endplate formation by the axons that have grown from these neurons of substitution throughout the nerve bridge, as well as the possibilities of reafferentiation of the transplanted tissues by regenerating host "central" nerve fibres, are in progress.     

Alteration of posttraumatic ischemia in experimental spinal cord trauma by a central nervous system depressant.

Blood flow after severe experimental injury to the thoracic spinal cord was studied in cats, using a modification of the hydrogen clearance technique. Gamma hydroxybutyrate, a central nervous system depressant, was shown to markedly alter the ischemic response to injury if given during the early posttraumatic period. Other vasoactive drugs investigated had no effect on posttraumatic ischemia. Therapeutic intervention during the early posttraumatic period aimed at increasing blood flow while decreasing the metabolic requirements of the injured cord may prove of value in reversing or limiting some elements of long-tract dysfunction due to the secondary ischemic insult.     

同种胚胎脊髓移植物对成年大鼠损伤脊髓组织形态与功能的修复作用

探讨大鼠胚胎脊髓组织移植物对成年大鼠损伤脊髓组织形态与功能的修复作用。在半切洞损伤的成年大鼠脊髓内，植入大鼠胚胎脊髓组织，术后行联合行为记分、诱发电位及组织学检查。结果：移植物能在宿主脊髓损伤部位存活，并能生长、分化、修复宿主脊髓的组织损伤，诱导宿生神经纤维联系的重建，改善宿主损伤脊髓的神经传导，促进功能恢复。以上结果提示：胚胎脊髓移植物对成年大鼠损伤脊髓组织形态与功能均具有修复作用。