Rat ventral caudal nerve as a model for long distance regeneration.

In the rat, tail nerves are the longest peripheral nerves in their body. We suggest that ventral caudal nerve (VCN) may serve as a model for studying nerve injury and long distance regeneration. For this purpose, we have studied the anatomy and morphometry of the VCN in control animals. 10 cm long segment of the VCN was removed, and transversal sections were collected at 10 mm distances. The myelinated axons were counted, and the series of data were used to characterize the craniocaudal tapering of the nerve. In a separate group of animals, retrograde tracing with Fluorogold was used to localize and quantitate the spinal neurons projecting their axons into the VCN. After complete nerve transection, the time course of histopathological changes in the distal segment was studied. The primary goal was to define the time needed for axonal disintegration. In later periods, axonal debris removal and rearrangement of tissue elements was documented. After compression injury (axonotmesis), Wallerian degeneration was followed by spontaneous regeneration of axons. We show that the growing axons will span the 10 cm distance within 4-8 weeks. After different survival periods, the numbers of regenerating axons were counted at 10 mm distances. These data were used to characterize the dynamics of axonal regeneration during 4 months' survival period. In the present study we show that axonal regeneration across 10 cm distance can be studied and quantitatively analyzed in a small laboratory animal.

Comparative model of minimal spinal cord injury reveals a rather anti-inflammatory response in the lesion site as well as increased proliferation in the central canal lining in the neonates compared to the adult rats.

Spinal cord injury (SCI) resulting from trauma decreases the quality of human life. Numerous clues indicate that the limited endogenous regenerative potential is a result of the interplay between the inhibitory nature of mature nervous tissue and the inflammatory actions of immune and glial cells. Knowledge gained from comparing regeneration in adult and juvenile animals could draw attention to factors that should be removed or added for effective therapy in adults. Therefore, we generated a minimal SCI (mSCI) model with a comparable impact on the spinal cord of Wistar rats during adulthood, preadolescence, and the neonatal period. The mechanism of injury is based on unilateral incision with a 20 ga needle tip according to stereotaxic coordinates into the dorsal horn of the L4 lumbar spinal segment. The incision should harm a similar amount of gray matter on a coronal section in each group of experimental animals. According to our results, the impact causes mild injury with minimal adverse effects on the neurological functions of animals but still has a remarkable effect on nervous tissue and its cellular and humoral components. Testing the mSCI model in adults, preadolescents, and neonates revealed a rather anti-inflammatory response of immune cells and astrocytes at the lesion site, as well as increased proliferation in the central canal lining in neonates compared with adult animals. Our results indicate that developing nervous tissue could possess superior reparative potential and confirm the importance of comparative studies to advance in the field of neuroregeneration.

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.

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.

The neuroprotective effect of rat adipose tissue-derived mesenchymal stem cell-conditioned medium on cortical neurons using an in vitro model of SCI inflammation.

Objectives In this study, a new approach was used with an in vitro model in which neural cells were exposed to conditioned media from the injured spinal cord (SCI-CM) mimicking a local inflammatory microenvironment . Subsequently, the neuroprotective effect of rat adipose tissue-derived msesenchymal stem cell-conditioned media (ATMSC-CM) was investigated through a cell-free based therapy, which was used to treat cortical neurons and astrocytes under inflammation. Methods Primary cell cultures isolated from postnatal day (P6) Wistar rat brain cortex were exposed to SCI-CM derived from the central lesion, rostral and caudal segments of injured spinal cord. After 48 h incubation, the SCI-CM was replaced and primary cultures were cultivated either in DMEM media alone or in ATMSC-CM for 72 h. The impact of ATMSC-CM on the viability of neurons and astrocytes was assessed using a CyQUANT® Direct Cell Proliferation Assay Kit as well as immunocytochemistry analysis. Results Immunocytochemical analysis revealed significant decrease in the number of MAP2 positive neurons exposed to SCI-CM compared to Control. Protection by ATMSC-CM was associated with increased survival of neurons compared to primary culture cultivated in DMEM media alone. The ATMSC-CM effect on astrocytes was more variable and without any significant impact. Conclusion The results demonstrate that SCI-CM mimicking inflammation can reduce cortical neuron survival, and subsequent exposure to ATMSC-CM can stabilize the neuronal population most likely via released neuroprotective and trophic factors. In addition, astrogliosis was not affected by ATMSC-CM.

RhoA Inhibitor Treatment At Acute Phase of Spinal Cord Injury May Induce Neurite Outgrowth and Synaptogenesis.

The therapeutic use of RhoA inhibitors (RhoAi) has been experimentally tested in spinal cord injury (SCI). In order to decipher the underlying molecular mechanisms involved in such a process, an in vitro neuroproteomic-systems biology platform was developed in which the pan-proteomic profile of the dorsal root ganglia (DRG) cell line ND7/23 DRG was assessed in a large array of culture conditions using RhoAi and/or conditioned media obtained from SCI ex vivo derived spinal cord slices. A fine mapping of the spatio-temporal molecular events of the RhoAi treatment in SCI was performed. The data obtained allow a better understanding of regeneration/degeneration induced above and below the lesion site. Results notably showed a time-dependent alteration of the transcription factors profile along with the synthesis of growth cone-related factors (receptors, ligands, and signaling pathways) in RhoAi treated DRG cells. Furthermore, we assessed in a rat SCI model the in vivo impact of RhoAi treatment administered in situ via alginate scaffold that was combined with FK506 delivery. The improved recovery of locomotion was detected only at the early postinjury time points, whereas after overall survival a dramatic increase of synaptic contacts on outgrowing neurites in affected segments was observed. We validate these results by in vivo proteomic studies along the spinal cord segments from tissue and secreted media analyses, confirming the increase of the synaptogenesis expression factors under RhoAi treatment. Taken together, we demonstrate that RhoAi treatment seems to be useful to stimulate neurite outgrowth in both in vitro as well in vivo environments. However, for in vivo experiments there is a need for sustained delivery regiment to facilitate axon regeneration and promote synaptic reconnections with appropriate target neurons also at chronic phase, which in turn may lead to higher assumption for functional improvement.

Axonal outgrowth stimulation after alginate/mesenchymal stem cell therapy in injured rat spinal cord.

Despite strong efforts in the field, spinal cord trauma still belongs among the untreatable neurological conditions at present. Given the complexity of the nervous system, an effective therapy leading to complete recovery has still not been found. One of the potential tools for supporting tissue regeneration may be found in mesenchymal stem cells, which possess anti­inflammatory and trophic factor­producing properties. In the context of transplantations, application of degradable biomaterials which could form a supportive environment and scaffold to bridge the lesion area represents another attractive strategy. In the present study, through a combination of these two approaches we applied both alginate hydrogel biomaterial alone or allogenic transplants of MSCs isolated from bone marrow seeded in alginate biomaterial into injured rat spinal cord at three weeks after spinal cord compression performed at Th8­9 level. Following three­week survival, using immunohistochemistry we studied axonal growth (GAP­43 expression) and both microglia (Iba­1) and astrocyte (GFAP) reactions at the lesion site and in the segments below and above the lesion. To detect functional improvement, during whole survival period we performed behavioral analyses of locomotor abilities using a classical open field test (BBB score) and a Catwalk automated gait analyzing device (Noldus). We found that despite the absence of locomotor improvement, application of both alginate and MSCs caused significant increase in the number of GAP­43 positive axons.

Proteomic Analysis of the Spatio-temporal Based Molecular Kinetics of Acute Spinal Cord Injury Identifies a Time- and Segment-specific Window for Effective Tissue Repair.

Spinal cord injury (SCI) represents a major debilitating health issue with a direct socioeconomic burden on the public and private sectors worldwide. Although several studies have been conducted to identify the molecular progression of injury sequel due from the lesion site, still the exact underlying mechanisms and pathways of injury development have not been fully elucidated. In this work, based on OMICs, 3D matrix-assisted laser desorption ionization (MALDI) imaging, cytokines arrays, confocal imaging we established for the first time that molecular and cellular processes occurring after SCI are altered between the lesion proximity, i.e. rostral and caudal segments nearby the lesion (R1-C1) whereas segments distant from R1-C1, i.e. R2-C2 and R3-C3 levels coexpressed factors implicated in neurogenesis. Delay in T regulators recruitment between R1 and C1 favor discrepancies between the two segments. This is also reinforced by presence of neurites outgrowth inhibitors in C1, absent in R1. Moreover, the presence of immunoglobulins (IgGs) in neurons at the lesion site at 3 days, validated by mass spectrometry, may present additional factor that contributes to limited regeneration. Treatment in vivo with anti-CD20 one hour after SCI did not improve locomotor function and decrease IgG expression. These results open the door of a novel view of the SCI treatment by considering the C1 as the therapeutic target.

Comparison of dynamic behavior and maturation of neural multipotent cells derived from different spinal cord developmental stages: an in vitro study.

Neural progenitor cells (NPCs) are characterized as undifferentiated cells with the ability of self-renewal and multipotency to give rise to other cells of the nervous system. In our in vitro study we demonstrate the proliferative and differentiative potential of NPCs isolated from the spinal cord at different developmental stages (embryonal, early postnatal, adult), maintained and expanded within neurospheres (NSs). Using the NSs culture system, we examined the size, number of NSs and their fate when exposed to differentiation conditions. Based on immunocytochemical analyses for cell markers (MAP 2, GFAP, RIP) we evaluated the occurrence of various cell types: neurons, astrocytes and oligodendrocytes. The results show that NSs increased in size during cultivation time via NPC proliferation, but proliferation potential decreased Turing maturation stages. In addition, NPCs derived from spinal cord developmentally different stages gave rise to a consistent ratio of glial and neuronal progeny (3:1), and adult tissues represent a comparable source of NPCs compared to embryonal and early postnatal tissues. These data provide useful information for large-scale in vitro expansion of NPCs required for potential cell therapy after spinal cord injury.

Modulation properties of factors released by bone marrow stromal cells on activated microglia: an in vitro study.

In the present paper we develop a new non-cell based (cell-free) therapeutic approach applied to BV2 microglial cells and spinal cord derived primary microglia (PM) using conditioned media from rat bone marrow stromal cells (BMSCs-CM). First we collected conditioned media (CM) from either naive or injured rat spinal cord tissue (SCI-CM, inflammatory stimulation agent) and from rat bone marrow stromal cells (BMSCs-CM, therapeutic immunomodulation agent). They were both subsequently checked for the presence of chemokines and growth, neurotrophic and neural migration factors using proteomics analysis. The data clearly showed that rat BMSCs-CM contain in vitro growth factors, neural migration factors, osteogenic factors, differentiating factors and immunomodulators, whereas SCI-CM contain chemokines, chemoattractant factors and neurotrophic factors. Afterwards we determined whether the BMSCs-CM affect chemotactic activity, NO production, morphological and pro-apoptotic changes of either BV2 or PM cells once activated with SCI-CM. Our results confirm the anti-migratory and NO-inhibitory effects of BMSCs-CM on SCI-CM-activated microglia with higher impact on primary microglia. The cytotoxic effect of BMSCs-CM occurred only on SCI-CM-stimulated BV2 cells and PM, not on naive BV2 cells, nor on PM. Taken together, the molecular cocktail found in BMSCs-CM is favorable for immunomodulatory properties.

Astrocytic and vascular scaffolding for neuroblast migration in the rostral migratory stream.

New neurons are continuously being added to the olfactory bulb (OB) of adult rodents that are generated in the subventricular zone (SVZ), distant by a few millimeters. Neuronal precursors have to overcome this long distance without the radial-glial migratory scaffold, in contrast to migration mode during embryonic development. The previous model explains migration of precursors from the SVZ through the rostral migratory stream (RMS) to the OB as a movement of neuroblasts along each other, ensheathed by astroglial tubes. Recent results indicate that blood vessels are suitable candidates for neuronal migration guidance in the RMS. These novel findings have changed the former concept accounting for neuronal precursor migration. The aim of our study was to map a pattern of vascularization in the RMS of adult rats and to investigate mutual relations among blood vessels, neuroblasts and astrocytes in this area. Detailed morphological analysis revealed that blood vessels in the RMS are organized in a specific manner. In most of the RMS extent, blood vessels run parallel to the outline of the migratory pathway. Interestingly, the caudal part of the RMS has a unique vasculature organization in which blood vessels create a spiral-like configuration. Chains of neuroblasts enveloped by astrocytes largely align along blood vessels. The exception is the caudal part of the RMS where neuroblasts do not follow non - parallel blood vessels. Our morphological findings suggest that blood vessels and astrocytes may cooperatively form physical substrate - scaffold for the neuroblasts migration in the RMS of adult rats.

A comparative study of PKH67, DiI, and BrdU labeling techniques for tracing rat mesenchymal stem cells.

Mesenchymal stem cells (MSCs) have generated a great deal of promise as a potential source of cells for cell-based therapies. Various labeling techniques have been developed to trace MSC survival, migration, and behavior in vitro or in vivo. In the present study, we labeled MSCs derived from rat bone marrow (rMSCs) with florescent membrane dyes PKH67 and DiI, and with nuclear labeling using 5 µM BrdU and 10 µM BrdU. The cells were then cultured for 6 d or passaged (1-3 passages). The viability of rMSCs, efficacy of fluorescent expression, and transfer of the dyes were assessed. Intense fluorescence in rMSCs was found immediately after membrane labeling (99.3 ± 1.6% PKH67+ and 98.4 ± 1.7% DiI+) or after 2 d when tracing of nuclei was applied (91.2 ± 4.6% 10 µM BrdU+ and 77.6 ± 4.6% 5 µM BrdU+), which remained high for 6 d. Viability of labeled cells was 91 ± 3.8% PKH67+, 90 ± 1.5% DiI+, 91 ± 0.8% 5 µM BrdU+, and 76.9 ± 0.9% 10 µM BrdU+. The number of labeled rMSCs gradually decreased during the passages, with almost no BrdU+ nuclei left at final passage 3. Direct cocultures of labeled rMSCs (PKH67+ or DiI+) with unlabeled rMSCs revealed almost no dye transfer from donor to unlabeled recipient cells. Our results confirm that labeling of rMSCs with PKH67 or DiI represents a non-toxic, highly stable, and efficient method suitable for steady tracing of cells, while BrdU tracing is more appropriate for temporary labeling due to decreasing signal over time.

Diverse effect of different odor stimuli on behavior and Fos protein production in the olfactory system neurogenic region of adult rats.

Previously it has been demonstrated that processes of postnatal neurogenesis in the olfactory system neurogenic region-the subventricular zone (SVZ), rostral migratory stream (RMS), and olfactory bulb (OB) can be significantly altered by different factors of an environment. However, the mechanisms involved in regulation of neurogenesis by exogenous factors in the olfactory system remain unclear. The purpose of the present study was to contribute to the understanding of these mechanisms by immunohistochemical assessment of Fos protein induction in areas of adult neurogenesis. To evaluate the coordinate activation of Fos production in neurons of the olfactory system neurogenic region, a brief exposure to artificial odor (eau de Cologne) or naturalistic odor (cat odor) has been used in alert rats. Our results revealed that the effects of these odors are easily distinguishable at both the behavioral and the morphological level. Cat odor induced greater changes in anxiety level, and produced typical pattern of Fos activation in the accessory olfactory bulb (AOB), a brain region associated with defensive behavior. An important finding is, that next to distinct Fos expression in the OB and the AOB, Fos positive cells have been found also within the SVZ/RMS of the odor stimulated rats. Interestingly, Fos expression in the RMS was detected only after exposure to artificial odor stimulus. These results provide new evidence that some SVZ/RMS cells have complete prerequisites necessary for the Fos signal transduction cascade.

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.

Effects of short-duration electromagnetic radiation on early postnatal neurogenesis in rats: Fos and NADPH-d histochemical studies.

The immediate effects of whole body electromagnetic radiation (EMR) were used to study postnatal neurogenesis in the subventricular zone (SVZ) and rostral migratory stream (RMS) of Wistar rats of both sexes. Newborn postnatal day 7 (P7) and young adult rats (P28) were exposed to pulsed electromagnetic fields (EMF) at a frequency of 2.45 GHz and mean power density of 2.8 mW/cm(2) for 2 h. Post-irradiation changes were studied using immunohistochemical localization of Fos and NADPH-d. We found that short-duration exposure induces increased Fos immunoreactivity selectively in cells of the SVZ of P7 and P28 rats. There were no Fos positive cells visible within the RMS of irradiated rats. These findings indicate that some differences exist in prerequisites of proliferating cells between the SVZ and RMS regardless of the age of the rats. Short-duration exposure also caused praecox maturation of NADPH-d positive cells within the RMS of P7 rats. The NADPH-d positive cells appeared several days earlier than in age-matched controls, and their number and morphology showed characteristics of adult rats. On the other hand, in the young adult P28 rats, EMR induced morphological signs typical of early postnatal age. These findings indicate that EMR causes age-related changes in the production of nitric oxide (NO), which may lead to different courses of the proliferation cascade in newborn and young adult neurogenesis.

Odor enrichment influences neurogenesis in the rostral migratory stream of young rats.

The olfactory bulb is one of a few brain structures characterized by high plasticity due to the fact that new neurons are continually integrated into the olfactory bulb circuit throughout life. The new cells originate from the subventricular zone of the forebrain and migrate through the rostral migratory stream (RMS) to the olfactory bulb that also represents the first synaptic relay of the olfactory system. Data accumulating in recent years have confirmed that sensory inputs can influence the level of postnatal neurogenesis in the olfactory bulb. In this study, we studied neurogenesis in the rostral migratory stream of Wistar albino rat pups after exposure to an odor-enriched environment. The rats were olfactory stimulated twice daily with different odorants from the day of their birth up to 1, 2 or 3 weeks, respectively. Using bromodeoxyuridine, a marker of cell proliferation, we found an increased number of proliferating cells in the rostral migratory stream of rat pups submitted to olfactory stimulation. Conversely, the number of dying cells, labeled with the fluorescent dye Fluoro Jade-C, was down-regulated in groups of rats exposed to an odor-enriched environment.