Characterisation of mesenchymal stem cells conditioned media obtained at different conditioning times: their effect on glial cells in in vitro scratch model.

In this study, the bone marrow mesenchymal stem cells conditioned media (BMMSC-CM) obtained by conditioning for 24(CM24), 48(CM48) and 72(CM72) hours was characterised. In vitro, the impact of BMMSC-CM on the astrocyte migratory response and oligodendrocyte density was evaluated using the scratch model. The proteomic profiles of individual secretomes were analysed by mass spectrometry and the concentrations of four selected neurotrophins (BDNF, NGF, GDNF and VEGF) were determined by ELISA. Our results revealed an increased number of proteins at CM72, many of which are involved in neuroregenerative processes. ELISA documented a gradual increase in the concentration of two neurotrophins (NGF, VEGF), peaking at CM72. In vitro, the different effect of individual BMMSC-CM on astrocyte migration response and oligodendrocyte density was observed, most pronounced with CM72. The outcomes demonstrate that the prolonged conditioning results in increased release of detectable proteins, neurotrophic factors concentration and stronger effect on reparative processes in neural cell cultures.

Prevention and therapy of chemotherapy-induced peripheral neuropathy: a review of recent findings.

Chemotherapy-induced peripheral neuropathy is one of the most frequent dose-limiting side effects, observed in patients receiving antineoplastic agents, persisting for up to two years after completing treatment, greatly affecting both the course of chemotherapy and patients' quality of life. Approximately 20 to 85% of patients treated with neurotoxic chemotherapy will develop peripheral neuropathy and there is considerable variability in its severity among patients. The main symptoms are numbness, paresthesia, and burning pain in a "glove and stocking" distribution. The prevalence of chemotherapy-induced peripheral neuropathy will likely increase as cancer survival rates continue to improve. Currently, there are only a few therapeutic options available for the prevention or successful therapy because the mechanisms of chemotherapy-induced peripheral neuropathy remain unclear. A better understanding of the risk factors and underlying mechanisms of chemotherapy-induced peripheral neuropathy is needed to develop effective preventive and therapeutic strategies.

Precision spinal gene delivery-induced functional switch in nociceptive neurons reverses neuropathic pain.

Second-order spinal cord excitatory neurons play a key role in spinal processing and transmission of pain signals to the brain. Exogenously induced change in developmentally imprinted excitatory neurotransmitter phenotypes of these neurons to inhibitory has not yet been achieved. Here, we use a subpial dorsal horn-targeted delivery of AAV (adeno-associated virus) vector(s) encoding GABA (gamma-aminobutyric acid) synthesizing-releasing inhibitory machinery in mice with neuropathic pain. Treated animals showed a progressive and complete reversal of neuropathic pain (tactile and brush-evoked pain behavior) that persisted for a minimum of 2.5 months post-treatment. The mechanism of this treatment effect results from the switch of excitatory to preferential inhibitory neurotransmitter phenotype in dorsal horn nociceptive neurons and a resulting increase in inhibitory activity in regional spinal circuitry after peripheral nociceptive stimulation. No detectable side effects (e.g., sedation, motor weakness, loss of normal sensation) were seen between 2 and 13 months post-treatment in naive adult mice, pigs, and non-human primates. The use of this treatment approach may represent a potent and safe treatment modality in patients suffering from spinal cord or peripheral nerve injury-induced neuropathic pain.

Formaldehyde-hardened albumin as a non-penetrating embedding matrix for frozen and vibratome sectioning.

In this paper, we describe a protocol for a non-penetrating embedding matrix that can be used for frozen or vibratome sectioning of various formaldehyde-fixed tissue specimens. In our experiments, we wanted to prepare thin frozen sections from miniature specimens for fluorescent staining. As we could not achieve satisfactory results with any of the previously published methods, we have tried to modify the existing protocols, and systematically evaluated the effect of these modifications on the properties of the embedding matrix. The resulting protocol is simple, the matrix gets firmly attached to the tissues, does not cause autofluorescence and enables preparing extremely thin frozen sections. The matrix can be used for 1, embedding miniature specimens from problematic tissues to enable cutting very thin frozen sections, 2, grouping multiple specimens into one large block for simultaneous processing, and 3, dispersing single cells and preparing cell blocks for frozen sectioning.

A method to prepare large resin sections for counting myelinated axons in rodent CNS and PNS structures.

We present a method that allows preparing histological sections from large blocks of nervous tissue embedded in epoxy resin. Resin-embedding provides excellent resolution especially for the myelin-rich white matter and is often being used for visualizing the myelinated axons in peripheral nerves. However, because of the limited penetration of the reagents, only very small tissue specimens can be processed in this way. Here, we describe a method that enables to embed large specimens and their sectioning on a standard sliding microtome. To process the large specimens, modifications in several steps of the processing technique had to be made. In this paper we demonstrate, that with this technique 1-3 µm thick transversal sections can be prepared from the resin-embedded specimens as large as rat brain hemisphere. Such a large section allows simultaneously: 1.) overviewing and delineating the gross anatomical structures, and 2.) observing the subcellular details at the highest possible optical magnifications. Such a large section with excellent resolution allows application of unbiased stereological methods and reliable quantification of very small objects within the area of interest.

Is Innervation of the Neuromuscular Junction at the Diaphragm Modulated by sGC/cGMP Signaling?

We previously reported NO/sGC signaling in the upper respiratory pathway, receiving input from the respiratory neurons of the brainstem to phrenic motoneurons in the C3-C6 spinal cord. In order to assess whether innervation of the neuromuscular junction (NMJ) at the diaphragm is modulated by sGC/cGMP signaling, we performed unilateral 8-day continuous ligation of the phrenic nerve in rats. We examined sGCß1 within the lower bulbospinal pathway (phrenic motoneurons, phrenic nerves and NMJs at the diaphragm) and the cGMP level in the contra- and ipsilateral hemidiaphragm. Additionally, we characterized the extent of phrenic nerve axonal degeneration and denervation at diaphragm NMJs. The results of our study show that continuous 8-day phrenic nerve ligation caused a marked increase in sGCß1 (immunoreactivity and the protein level) in the ipsilateral phrenic motor pool. However, the protein sGCß1 level in the phrenic nerve below its ligation and the cGMP level in the ipsilateral hemidiaphragm were evidently decreased. Using confocal analysis we discovered a reduction in sGCß1-IR boutons/synaptic vesicles at the ipsilateral MNJs. These findings are consistent with the marked axonal loss (∼47%) and significant NMJs degeneration in the ipsilateral diaphragm muscle. The remarkable unilateral decrease in cGMP level in the diaphragm and the failure of EMG recordings in the ipsilateral hemidiaphragm muscle can be attributed to the fact that sGC is involved in transmitter release at the diaphragm NMJs via the sGC-cGMP pathway.

Spinal subpial delivery of AAV9 enables widespread gene silencing and blocks motoneuron degeneration in ALS.

Gene silencing with virally delivered shRNA represents a promising approach for treatment of inherited neurodegenerative disorders. In the present study we develop a subpial technique, which we show in adult animals successfully delivers adeno-associated virus (AAV) throughout the cervical, thoracic and lumbar spinal cord, as well as brain motor centers. One-time injection at cervical and lumbar levels just before disease onset in mice expressing a familial amyotrophic lateral sclerosis (ALS)-causing mutant SOD1 produces long-term suppression of motoneuron disease, including near-complete preservation of spinal α-motoneurons and muscle innervation. Treatment after disease onset potently blocks progression of disease and further α-motoneuron degeneration. A single subpial AAV9 injection in adult pigs or non-human primates using a newly designed device produces homogeneous delivery throughout the cervical spinal cord white and gray matter and brain motor centers. Thus, spinal subpial delivery in adult animals is highly effective for AAV-mediated gene delivery throughout the spinal cord and supraspinal motor centers.

In vitro predegeneration of peripheral nerve; the effect of predegeneration period on rat Schwann cell cultures.

Peripheral nerve predegeneration has been used as a tool to improve the in vitro cultivation of Schwann cells. The process of predegeneration may be accomplished either in vivo or in vitro. In previously published studies, various predegeneration periods were used, ranging from a few days until up to 5 weeks. The present study systematically evaluated the effect of various durations of in vitro predegeneration on the efficacy of Schwann cell cultivation. The sciatic nerves of adult Wistar rats were harvested and the explanted nerve pieces were maintained in the predegeneration medium for different predegeneration periods. In group A, the dissociation was performed immediately after harvesting. In groups B, C and D, the predegeneration periods were 2, 4 and 6 weeks, respectively. During the predegeneration period, the tissue pieces were repeatedly transferred into new dishes. Afterwards, the nerve tissue was enzymatically dissociated and the cells were seeded onto a six-well culture plate at a defined density. After 3-4 days of incubation, the cultures were passaged by means of the cold jet technique and the cell cultivation was continued for another 21 days. It was observed that the cell cultures in groups A and B were rapidly overgrown by fibroblasts. In group C, numerous wells contained a highly enriched Schwann cell population that had formed a typical monolayer, but in a fraction of the dishes, cultures were debased by fibroblast overgrowth. In group D, all of the cultures had enriched Schwann cell populations. In the experiments of the present study, the positive effect of predegeneration was observed only when the predegeneration periods lasted for 4 weeks or longer. It was concluded that the longer predegeneration periods activated Schwann cells and/or depleted the fibroblast proliferation capacity.

Immunosuppressant FK506: focusing on neuroprotective effects following brain and spinal cord injury.

The secondary damage that follows central nervous system (CNS) injury is a target for neuroprotective agents aimed at tissue and function sparing. FK506, a clinically used immunosuppressant, acts neuroprotectively in rat models of brain and spinal cord injury and ischemia. Evidence of in vivo experimental studies highlights the neuroprotective role of FK506 by its direct impact on various cell populations within the CNS. The participation of FK506 in modulation of post-traumatic inflammatory processes is a further potential aspect involved in CNS neuroprotection. In this review we provide an overview of the current laboratory research focusing on the multiple effects of FK506 on neuroprotection following CNS injury.

Regenerative medicine for the treatment of spinal cord injury: more than just promises?

Spinal cord injury triggers a complex set of events that lead to tissue healing without the restoration of normal function due to the poor regenerative capacity of the spinal cord. Nevertheless, current knowledge about the intrinsic regenerative ability of central nervous system axons, when in a supportive environment, has made the prospect of treating spinal cord injury a reality. Among the range of strategies under investigation, cell-based therapies offer the most promising results, due to the multifactorial roles that these cells can fulfil. However, the best cell source is still a matter of debate, as are clinical issues that include the optimal cell dose as well as the timing and route of administration. In this context, the role of biomaterials is gaining importance. These can not only act as vehicles for the administered cells but also, in the case of chronic lesions, can be used to fill the permanent cyst, thus creating a more favourable and conducive environment for axonal regeneration in addition to serving as local delivery systems of therapeutic agents to improve the regenerative milieu. Some of the candidate molecules for the future are discussed in view of the knowledge derived from studying the mechanisms that facilitate the intrinsic regenerative capacity of central nervous system neurons. The future challenge for the multidisciplinary teams working in the field is to translate the knowledge acquired in basic research into effective combinatorial therapies to be applied in the clinic.

Repetitive intrathecal catheter delivery of bone marrow mesenchymal stromal cells improves functional recovery in a rat model of contusive spinal cord injury.

Transplantation of bone marrow mesenchymal stromal cells (MSCs) has been shown to improve the functional recovery in various models of spinal cord injury (SCI). However, the issues of the optimal dose, timing, and route of MSC application are crucial factors in achieving beneficial therapeutic outcomes. The objective of this study was to standardize the intrathecal (IT) catheter delivery of rat MSCs after SCI in adult rats. MSCs labeled with PKH-67 were administered by IT delivery to rats at 3 or 7 days after SCI as one of the following treatment regimens: (1) a single injection (5×10(5) MSCs/rat), or (2) as three daily injections (5×10(5) MSCs/rat/d for a total of 1.5×10(6) MSCs/rat over 3 days, injected on days 3, 4, and 5, or days 7, 8, and 9 following SCI. The animals were behaviorally tested for 4 weeks using the Basso, Beattie, and Bresnahan (BBB) locomotor rating scale, and histologically assessed for MSC survival, distribution, and engraftment properties after 28 days. Rats treated with a single injection of MSCs at 3 or 7 days post-injury showed a modest, non-significant improvement in function and low survival of grafted MSCs, which were found attached to the pia mater or accumulated around the anterior spinal artery. In contrast, rats treated with three daily injections of MSCs at days 7, 8, and 9, but not on days 3, 4, and 5, showed significantly higher motor function recovery (BBB score 16.8±1.7) at 14-28 days post-injury. Transplanted PKH-67 MSCs were able to migrate and incorporate into the central lesion. However, only a limited number of surviving MSCs, ranging from 24,128±1170 to 116,258±8568 cells per graft, were observed within the damaged white matter. These results suggest that repetitive IT transplantation, which imposes a minimal burden on the animals, may improve behavioral function when the dose, timing, and targeted IT delivery of MSCs towards the lesion cavity are optimized.

NMDA receptors are expressed in lymphocytes activated both in vitro and in vivo.

There is increasing evidence showing that the interplay between neuronal and immune systems may be regulated by neuromediators. However, little is known about the involvement of glutamatergic system in such neuro-immune relations. In the present study, we have shown that some intact lymphocytes express N-methyl-D: -aspartate activated receptors (NMDA receptors), an important constituent of glutamatergic system. The activation of lymphocytes with phytohemagglutinin (PHA) induces a time-dependent increase in the amount of NMDA receptor presenting cells, and NMDA stimulates this process. Immune response of such lymphocytes is suppressed and the amount of cells producing interferon gamma (IFN-gamma) in vitro is decreased to the level corresponding to intact (non-activated) cells. Furthermore, lymphocytes in the region of inflammation, induced by spinal cord injury (SCI), are also NMDA-positive. We suggest that expression of NMDA receptors in lymphocytes is regulated by central nervous system, which controls the inflammation process.

Effects of long-term FK506 administration on functional and histopathological outcome after spinal cord injury in adult rat.

FK506 (tacrolimus), a potent immunosuppressive drug primarily used for reduction of allograft rejection in organ transplantation, also offers neuroprotection after central nervous system injury. FK506-mediated immunosuppression and neuroprotection may occur through different mechanisms that could affect neurological recovery and the severity of spinal lesions where cells transplantation therapy is combined with FK506 application. We assessed effects of long-term FK506 administration using the same dose regiment (1 mg/kg/day for 6 weeks) as is used in spinal cord transplantation studies following a balloon-compression induced spinal cord injury (SCI). Body weight and locomotor recovery quantified by the BBB (Basso-Beattie-Bresnehan) locomotor rating scale were evaluated for up to 42 days post-injury. The area of the preserved spinal cord tissue within a 13 mm segment of the spinal cord (lesion epicenter and 6 mm rostral-caudal) was examined histologically. The results showed no significant effects of FK506 on spinal cord tissue sparing or improvement of locomotor recovery. However, body weight fell significantly (P < 0.05) with FK506 treatment when compared with placebo from day 7 until sacrifice. In our experimental design, long-term FK506 treatment did not affect the parameters of outcome following balloon-compression SCI in the rat; however, multiple effects of FK506 should be taken into account when evaluating the outcomes in transplantation studies.

Response of ependymal progenitors to spinal cord injury or enhanced physical activity in adult rat.

Ependymal cells (EC) in the spinal cord central canal (CC) are believed to be responsible for the postnatal neurogenesis following pathological or stimulatory conditions. In this study, we have analyzed the proliferation of the CC ependymal progenitors in adult rats processed to compression SCI or enhanced physical activity. To label dividing cells, a single daily injection of Bromo-deoxyuridine (BrdU) was administered over a 14-day-survival period. Systematic quantification of BrdU-positive ependymal progenitors was performed by using stereological principles of systematic, random sampling, and optical Dissector software. The number of proliferating BrdU-labeled EC increased gradually with the time of survival after both paradigms, spinal cord injury, or increased physical activity. In the spinal cord injury group, we have found 4.9-fold (4 days), 7.1-fold (7 days), 4.9-fold (10 days), and 5.6-fold (14 days) increase of proliferating EC in the rostro-caudal regions, 4 mm away from the epicenter. In the second group subjected to enhanced physical activity by running wheel, we have observed 2.1-2.6 fold increase of dividing EC in the thoracic spinal cord segments at 4 and 7 days, but no significant progression at 10-14 days. Nestin was rapidly induced in the ependymal cells of the CC by 2-4 days and expression decreased by 7-14 days post-injury. Double immunohistochemistry showed that dividing cells adjacent to CC expressed astrocytic (GFAP, S100beta) or nestin markers at 14 days. These data demonstrate that SCI or enhanced physical activity in adult rats induces an endogenous ependymal cell response leading to increased proliferation and differentiation primarily into macroglia or cells with nestin phenotype.

Cortical and hippocampal neurons from truncated tau transgenic rat express multiple markers of neurodegeneration.

Transition of protein tau from physiologically unfolded to misfolded state represent enigmatic step in the pathogenesis of tauopathies including Alzheimer's disease (AD). Major molecular events playing role in this process involve truncation and hyperphosphorylation of tau protein, which are accompanied by redox imbalance followed by functional deterioration of neuronal network. Recently we have developed transgenic rat model showing that expression of truncated tau causes neurofibrillary degeneration similar to that observed in brain of AD sufferers. Consequently we tested cortical and hippocampal neuronal cultures extracted from this model as a convenient tool for development of molecules able to target the mechanisms leading to and/or enhancing the process of neurodegeneration. Here we document three major pathological features typical for tauopathies and AD in cortical and hippocampal neurons from transgenic rat in vitro. First, an increased accumulation of human truncated tau in neurons; second, the hyperphosphorylation of truncated tau on the epitopes characteristic of AD (Ser202/Thr205 and Thr231); and third, increased vulnerability of the neurons to nitrative and oxidative stress. Our results show that primary neurons expressing human truncated tau could represent a cellular model for targeting tau related pathological events, namely, aberrant tau protein accumulation, tau hyperphosphorylation, and oxidative/nitrative damage. These characteristics make the model particularly suitable for detailed study of molecular mechanisms of tau induced neurodegeneration and easily applicable for drug screening.

NeuroScale, the battery of behavioral tests with novel scoring system for phenotyping of transgenic rat model of tauopathy.

We have previously shown that transgenic rats expressing misfolded tau protein developed neurofibrillary tangles and axonal degeneration in the brain and spinal cord, which led to impairment of sensorimotor and neuromuscular functions. To quantify neurobehavioral phenotype of the transgenic rats we have designed a testing protocol and a novel scoring system - NeuroScale - that reliably reflects progression of functional impairment of transgenic rats. NeuroScale consists of three variants of beam walking test with different sensitivity and a rapid neuromuscular and neurological examination, where animal performance is evaluated and scored according to a pre-defined rating scale. The range of the rating scale was developed to increase homogeneity of the collected behavioral data without lowering sensitivity of the testing methods. Finally, all awarded points were summed up to obtain a complete quantitative behavioral readout, the NeuroScale score, from animals under investigation. Increase in the NeuroScale score faithfully mirrored disease progression and allowed statistically significant discrimination (p<0.001) between behavioral responses of transgenic and control animals during the whole disease process. The method was suitable for a high-throughput test whereby an experienced operator can examine up to 60 rats per day. We show that this multi-test battery with novel sensitive scoring system - NeuroScale - represents a rapid, simple to perform, high throughput method for quantitative evaluation of behavioral phenotype of transgenic rats that could serve as a valuable primary read-out for in vivo validation of therapeutic agents.

Local transcutaneous cooling of the spinal cord in the rat: effects on long-term outcomes after compression spinal cord injury.

This study tested efficiency of a novel thermoelectric cooler for local transcutaneous spinal cord cooling. Spinal cord compression was made by epidural balloon inflation at T8-T9 level of the spinal cord. Experimental animals (n=20) were divided into two groups. In the hypothermic group, local cooling started 25 min after spinal cord injury and lasted for 1 h with paravertebral temperature maintained at 28.5 degrees C (+/-0.3). Normothermic group underwent identical procedures, but their temperature was maintained normothermic. The assessment of neurologic recovery was performed once a week during a 4 weeks survival period. After 4 weeks animals were sacrificed and the extent of the spinal cord lesion morphometrically evaluated. There were no statistically significant intergroup differences in BBB scores and preserved volumes of the spinal cord tissue. In consecutive cross-sectional areas, hypothermic animals had significantly more preserved white matter at the cranial periphery of the lesion. It was concluded that mild posttraumatic hypothermia (31.8 degrees C) had some protective effect on tissue loss after spinal cord injury but this effect was not associated with functional improvement.

Limited minocycline neuroprotection after balloon-compression spinal cord injury in the rat.

Minocycline (MC), a second-generation tetracycline and anti-inflammatory agent reportedly provides neuroprotection following CNS injury. The objective of this study was to examine the neuroprotective effects of short and long-term MC treatment using balloon-compression spinal cord injury (SCI) in the rat. Rats subjected to SCI were treated with MC for 1 day (1DMC group; total dose 180 mg/kg) or 5 days (5DMC group; total dose 450 mg/kg) or placebo. The effects of MC treatment on locomotor recovery (BBB scale) and spinal cord white and gray matter sparing were evaluated for up to 28 days. Morphometric analysis showed that while MC treatment spared spinal cord white and gray matter rostral to the lesion epicenter in both, 1DMC and 5DMC groups, sparing of white and gray matter areas was not observed caudal to the traumatic lesion. In addition, MC treatment had no effect on final locomotor recovery. Limited improvement of spinal cord post-compression consequences raises questions about the neuroprotection efficiency of MC treatment following compression SCI in the rat.

Traumatic injury of the spinal cord and nitric oxide.

In the current report, we summarize our findings related to the involvement of nitric oxide (NO) in the pathology of spinal cord trauma. We initially studied the distribution of nitric oxide synthase (NOS)-immunolabeled and/or nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd; which is highly colocalized with NOS)-stained somata and fibers in the spinal cord of the rabbit. Segmental and laminar distribution of NADPHd-stained neurons in the rabbit revealed a large number of NADPHd-stained neurons in the spinal cord falling into six categories, N1-N6, while others could not be classified. Large numbers of NADPHd-stained neurons were identified in the superficial dorsal horn and around the central canal. Four morphologically distinct kinds of NADPHd-stained axons 2.5-3.5 microm in diameter were identified throughout the white matter in the spinal cord. Moreover, a massive occurrence of axonal NADPHd-staining was detected in the juxtagriseal layer of the ventral funiculus along the rostrocaudal axis. The prominent NADPHd-stained fiber bundles were identified in the mediobasal and central portion of the ventral funiculus. The sulcomarginal fasciculus was found in the basal and medial portion of the ventral funiculus in all cervical and thoracic segments. Since the discovery that NO may act as a neuronal transmitter, an increasing interest has focused on its ability to modulate synaptic function. NO passes through cell membranes without specific release or uptake mechanisms inducing changes in signal-related functions by several means. In particular, the activation of the soluble guanylyl cyclases (sGC), the formation of cyclic guanosine 3',5'-monophosphate (cGMP) and the action of cGMP-dependent protein kinases has been identified as the main signal transduction pathways of NO in the nervous system including spinal cord. It is known that the intracellular level of cGMP is strictly controlled by its rate of synthesis via guanylyl cyclases (GC) and/or by the rate of its degradation via 3',5'-cyclic nucleotide phosphodiesterases (PDE). GC can be divided into two main groups, i.e., the membrane-bound or particular guanylyl cyclase (pGC) and the cytosolic or sGC. In the spinal cord, the activation of pGC has only been demonstrated for natriuretic peptides, which stimulate cGMP accumulation in GABA-ergic structures in laminae I-III of the rat cervical spinal cord. These neurons are involved in controlling the action of the locomotor circuit. In view of the abundance of NO-responsive structures in the brain, it is proposed that NO-cGMP signaling will be part of neuronal information processing at many levels. In relation to this, we found that surgically induced Th7 constriction of 24 h duration stimulated both the constitutive NOS activity and cGMP level by 120 and 131%, respectively, in non-compartmentalized white matter of Th8-Th9 segments, located just caudally to the site of injury. NO-mediated cGMP formation was only slightly increased in the dorsal funiculus of Th5-Th9 segments. There are some other sources that may influence the NO-mediated cGMP formation in spinal cord. A high level of glutamate produced at the site of the lesion and an excessive accumulation of intracellular Ca2+ may stimulate NOS activity and create suitable conditions for NO synthesis and its adverse effect on white matter. An increased interest has focused on the role of NO at the site of injury and in areas located close to the epicenter of the impact site and, in these connections an upregulation of NOS was noted in neurons and interneurons. However, the upregulation of NOS expression was also seen in interneurons located just rostrally and caudally to the lesion. A quantitative analysis of laminar distribution of multiple cauda equina constriction (MCEC) induced NADPHd-stained neurons revealed a considerable increase in these neurons in laminae VIII-IX 8h postconstriction, and a highly statistically significant increase of such neurons in laminae VII-X 5 days postconstriction in the lumbosacral segments. Concurrently, the number of NADPHd-stained neurons on laminae I-II in LS segments was greatly reduced. It is concluded that a greater understanding of NO changes after spinal cord trauma is essential for the possibility of targeting this pathway therapeutically.

Transplants of human mesenchymal stem cells improve functional recovery after spinal cord injury in the rat.

Human mesenchymal stem cells (hMSCs) derived from adult bone marrow represent a potentially useful source of cells for cell replacement therapy after nervous tissue damage. They can be expanded in culture and reintroduced into patients as autografts or allografts with unique immunologic properties. The aim of the present study was to investigate (i) survival, migration, differentiation properties of hMSCs transplanted into non-immunosuppressed rats after spinal cord injury (SCI) and (ii) impact of hMSC transplantation on functional recovery. Seven days after SCI, rats received i.v. injection of hMSCs (2x10(6) in 0.5 mL DMEM) isolated from adult healthy donors. Functional recovery was assessed by Basso-Beattie-Bresnahan (BBB) score weekly for 28 days. Our results showed gradual improvement of locomotor function in transplanted rats with statistically significant differences at 21 and 28 days. Immunocytochemical analysis using human nuclei (NUMA) and BrdU antibodies confirmed survival and migration of hMSCs into the injury site. Transplanted cells were found to infiltrate mainly into the ventrolateral white matter tracts, spreading also to adjacent segments located rostro-caudaly to the injury epicenter. In double-stained preparations, hMSCs were found to differentiate into oligodendrocytes (APC), but not into cells expressing neuronal markers (NeuN). Accumulation of GAP-43 regrowing axons within damaged white matter tracts after transplantation was observed. Our findings indicate that hMSCs may facilitate recovery from spinal cord injury by remyelinating spared white matter tracts and/or by enhancing axonal growth. In addition, low immunogenicity of hMSCs was confirmed by survival of donor cells without immunosuppressive treatment.