Hypothermia in the course of acute traumatic spinal cord injury.

In this review we briefly discuss animal experiments involving acute traumatic spinal cord injury (SCI) and the need for larger animals in testing experimental therapies. This literature overview, including the discussion of our own results from animal models, examines the use of hypothermia as a treatment method for SCI. Finally, we report the results of hypothermia application in clinical trials. Minipigs have been proposed as a potentially preferable model to rodents (typically rats) for predicting outcomes in human SCI due to their closer anatomical similarity to humans. In various animal studies, hypothermic treatment applied in the acute phase after SCI has resulted in neuroprotective effects, most likely due to inhibition of blood flow and oxygen consumption and reduction of overall metabolic activity and inflammation, resulting in improved nerve tissue sparing. Small­scale human clinical trials have been carried out, involving general (whole­body, systemic) or local hypothermia (close to the SCI site), with encouraging results. Nevertheless, further multi­center, randomized, double­blind studies with much larger patient numbers are necessary so that protocols can be standardized in order for hypothermia treatment to be reliably applied in clinical practice .

Diffusion Tensor Imaging: Tool for Tracking Injured Spinal Cord Fibres in Rat.

Spinal cord injury (SCI) is a severe disorder of the CNS leading to tissue damage and disability. Because it is critical to understand the pathological processes, it is important to find efficient ways to diagnose the severity of injured spinal cord tracts in situ from beginning up to a certain level of recovery following therapeutic interventions. In the current study, we set-up the criteria for diffusion tensor imaging (DTI) in order to capture changes of nerve fibre tracts in rat spinal cord compression injury. We tested four DTI parameters, such as fractional anisotropy, mean diffusivity, axial diffusivity and radial diffusivity at the lesion site, in time course of 7 weeks. Afterwards, we compared DTI data with histological results and locomotor outcomes to examine their consistency and capability of reflecting the lesion development in time. Our data confirm that DTI is a valuable in vivo imaging tool capable to distinguish damaged white matter tracts after mild SCI in rat. Fractional anisotropy showed decreased values for injury site, while the mean diffusivity had higher values, with increased both axial and radial diffusivity in comparison to control subjects. Thus, the combination of DTI parameters can reflect the severity of lesion in time and may correlate with histological evaluation of spared tissue, but not with locomotor recovery following mild injury associated with spontaneous recovery.

Hypothermic treatment after computer-controlled compression in minipig: A preliminary report on the effect of epidural vs. direct spinal cord cooling.

The aim of the present study was to investigate the therapeutic efficacy of local hypothermia (beginning 30 min post-injury persisting for 5 h) on tissue preservation along the rostro-caudal axis of the spinal cord (3 cm cranially and caudally from the lesion site), and the prevention of injury-induced functional loss in a newly developed computer-controlled compression model in minipig (force of impact 18N at L3 level), which mimics severe spinal cord injury (SCI). Minipigs underwent SCI with two post-injury modifications (durotomy vs. intact dura mater) followed by hypothermia through a perfusion chamber with cold (epidural t≈15°C) saline, DMEM/F12 or enriched DMEM/F12 (SCI/durotomy group) and with room temperature (t≈24°C) saline (SCI-only group). Minipigs treated with post-SCI durotomy demonstrated slower development of spontaneous neurological improvement at the early postinjury time points, although the outcome at 9 weeks of survival did not differ significantly between the two SCI groups. Hypothermia with saline (t≈15°C) applied after SCI-durotomy improved white matter integrity in the dorsal and lateral columns in almost all rostro-caudal segments, whereas treatment with medium/enriched medium affected white matter integrity only in the rostral segments. Furthermore, regeneration of neurofilaments in the spinal cord after SCI-durotomy and hypothermic treatments indicated an important role of local saline hypothermia in the functional outcome. Although saline hypothermia (24°C) in the SCI-only group exhibited a profound histological outcome (regarding the gray and white matter integrity and the number of motoneurons) and neurofilament protection in general, none of the tested treatments resulted in significant improvement of neurological status. The findings suggest that clinically-proven medical treatments for SCI combined with early 5 h-long saline hypothermia treatment without opening the dural sac could be more beneficial for tissue preservation and neurological outcome compared with hypothermia applied after durotomy.

Survival of syngeneic and allogeneic iPSC-derived neural precursors after spinal grafting in minipigs.

The use of autologous (or syngeneic) cells derived from induced pluripotent stem cells (iPSCs) holds great promise for future clinical use in a wide range of diseases and injuries. It is expected that cell replacement therapies using autologous cells would forego the need for immunosuppression, otherwise required in allogeneic transplantations. However, recent studies have shown the unexpected immune rejection of undifferentiated autologous mouse iPSCs after transplantation. Whether similar immunogenic properties are maintained in iPSC-derived lineage-committed cells (such as neural precursors) is relatively unknown. We demonstrate that syngeneic porcine iPSC-derived neural precursor cell (NPC) transplantation to the spinal cord in the absence of immunosuppression is associated with long-term survival and neuronal and glial differentiation. No tumor formation was noted. Similar cell engraftment and differentiation were shown in spinally injured transiently immunosuppressed swine leukocyte antigen (SLA)-mismatched allogeneic pigs. These data demonstrate that iPSC-NPCs can be grafted into syngeneic recipients in the absence of immunosuppression and that temporary immunosuppression is sufficient to induce long-term immune tolerance after NPC engraftment into spinally injured allogeneic recipients. Collectively, our results show that iPSC-NPCs represent an alternative source of transplantable NPCs for the treatment of a variety of disorders affecting the spinal cord, including trauma, ischemia, or amyotrophic lateral sclerosis.

Neuroprotective effect of local hypothermia in a computer-controlled compression model in minipig: Correlation of tissue sparing along the rostro-caudal axis with neurological outcome.

This study investigated the neuroprotective efficacy of local hypothermia in a minipig model of spinal cord injury (SCI) induced by a computer-controlled impactor device. The tissue integrity observed at the injury epicenter, and up to 3 cm cranially and caudally from the lesion site correlated with motor function. A computer-controlled device produced contusion lesions at L3 level with two different degrees of tissue sparing, depending upon pre-set impact parameters (8N- and 15N-force impact). Hypothermia with cold (4°C) saline or Dulbecco's modified Eagle's medium (DMEM)/F12 culture medium was applied 30 min after SCI (for 5 h) via a perfusion chamber (flow 2 ml/min). After saline hypothermia, the 8N-SCI group achieved faster recovery of hind limb function and the ability to walk from one to three steps at nine weeks in comparison with non-treated animals. Such improvements were not observed in saline-treated animals subjected to more severe 15N-SCI or in the group treated with DMEM/F12 medium. It was demonstrated that the tissue preservation in the cranial and caudal segments immediately adjacent to the lesion, and neurofilament protection in the lateral columns may be essential for modulation of the key spinal microcircuits leading to a functional outcome. Tissue sparing observed only in the caudal sections, even though significant, was not sufficient for functional improvement in the 15N-SCI model.

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.

The effect of cauda equina constriction on nitric oxide synthase activity.

Nitric oxide synthase (NOS) activity was studied in the gray and white matter regions of the spinal cord 2 and 5 days after multiple cauda equina constrictions of the central processes of L7-Co5 dorsal root ganglia neurons. The results show considerable differences in enzyme activity in the thoracic, upper lumbar, lower lumbar, and sacral segments. Increased NOS activity was observed at 2 days after multiple cauda equina constrictions in the dorsal, lateral, and ventral columns of the lower lumbar segments and in the ventral column of the upper lumbar segments. The values returned to control levels within 5 postconstriction days. In the lateral columns of thoracic segments taken 2 and 5 days after surgery, NOS activity was enhanced by 54% and 55% and in the upper lumbar segments by 130% and 163%, respectively. Multiple cauda equina constrictions performed surgically for 2 and 5 days caused a significant increase in NOS activity predominantly in the gray matter regions of thoracic segments. A quite different response was found 5 days postconstriction in the upper lumbar segments, where the enzyme activity was significantly decreased in the dorsal horn, intermediate zone, and ventral horn. No such extreme differences could be seen in the lower lumbar segments, where NOS activity was significantly enhanced only in the ventral horn. The data correspond with a higher number of NOS immunoreactive somata, quantitatively evaluated in the ventral horn of the lower lumbar segments at 5 days after multiple cauda equina constrictions. While the great region-dependent heterogeneity in NOS activity seen 2 and 5 days after multiple cauda equina constrictions is quite apparent and suggestive of an active role played by nitric oxide in neuroprotective or neurotoxic processes occurring in the gray and white matter of the spinal cord, the extent of damage or the degree of neuroprotection caused by nitric oxide in compartmentalized gray and white matter in this experimental paradigm would be possible only using longer postconstriction periods.

The case for the bulbospinal respiratory nitric oxide synthase-immunoreactive pathway in the dog.

Previous investigations from our laboratory have documented that the neuropil of the phrenic nucleus contains a dense accumulation of punctate nicotinamide adenine dinucleotide phosphate diaphorase staining. In this study we investigated the occurrence and origin of punctate nitric oxide synthase immunoreactivity in the neuropil of the phrenic nucleus in C3-C5 segments, supposed to be the terminal field of the premotor bulbospinal respiratory nitric oxide synthase-immunoreactive pathway in the dog. As the first step, nitric oxide synthase immunohistochemistry was used to characterize nitric oxide synthase-immunoreactive staining of the phrenic nucleus and nitric oxide synthase-containing neurons in the dorsal and rostral ventral respiratory group and in the Bötzinger complex of the medulla. Dense punctate nitric oxide synthase immunoreactivity was found on control sections in the neuropil of the phrenic nucleus. Several thin bundles of nitric oxide synthase-immunoreactive fibers were found to enter the phrenic nucleus from the lateral and ventral column. Nitric oxide synthase-containing neurons were revealed in the dorsal respiratory group of medulla corresponding to the ventrolateral nucleus of the solitary tract and in the rostral ventral respiratory group beginning approximately 1 mm caudal to the obex and reaching to 650 microm rostral to the obex. Axotomy-induced retrograde changes, consisting in a strong upregulation of nitric oxide synthase-containing neurons, were found in the dorsal and rostral ventral respiratory group contralateral to the hemisection performed at the C2-C3 level. Concurrently, a strong depletion of the punctate nitric oxide synthase immunopositivity in the neuropil of the phrenic nucleus ipsilaterally with the hemisection was detected, thus revealing that a crossed premotor bulbospinal respiratory pathway contains a fairly high number of nitric oxide synthase-immunopositive fibers terminating in the phrenic nucleus. The use of the retrograde fluorescent tracer Fluorogold injected into the phrenic nucleus and an analysis of sections cut through the dorsal and rostral ventral respiratory group and Bötzinger complex of medulla and processed for nitric oxide synthase immunocytochemistry revealed that approximately 73.8% of crossed premotor bulbospinal respiratory nitric oxide synthase-immunoreactive axons originate in the rostral ventral respiratory group and 26.2% is given by nitric oxide synthase-containing neurons of the dorsal respiratory group. A few premotor nitric oxide synthase-immunoreactive axons originating from the Bötzinger complex were found. In summary, the present study provides evidence for a hitherto unknown premotor bulbospinal respiratory nitric oxide synthase-immunoreactive pathway connecting the bulbar respiratory centers with the motor neurons of the phrenic nucleus in the dog.