Low Fos expression in newly generated neurons of the main and accessory olfactory bulb following single maternal separation.

The main and accessory olfactory bulbs (MOB and AOB) are unique in that they produce new neurons throughout adulthood. Despite the recent knowledge about the involvement of postnatally generated cells in several aspects of olfaction, the functional role of these neurons is still not sufficiently understood. The function of newly generated olfactory bulb neurons is primarily investigated in relation to activities related to smell. Stress-induced activation of new olfactory neurons has not yet been studied. Thus, our work was aimed to investigate whether a stressful event, such as maternal separation (MS) can induce Fos expression in postnatally-born neurons in the MOB and AOB. Rat pups were exposed to single maternal separation (SMS) for 2 h at the postnatal days: P7, P14, and P21. Quantification of immunohistochemically labeled Fos + cells revealed that exposure to SMS in different age stages during the first postnatal month stimulates activity in cells of individual MOB/AOB layers in an age-dependent manner. In order to find out whether newly generated cells in the MOB/AOB could express Fos protein as a response to SMS, newborn rats were administrated with the marker of proliferation, bromodeoxyuridine (BrdU) at P0, and three weeks later (at P21) colocalization of Fos and BrdU in the neurons of the MOB and AOB was assessed. Quantitative analysis of BrdU/Fos double-labeled cells showed that Fos is expressed only in a small number of postnatally generated cells within the MOB/AOB. Our results indicate that postnatally generated MOB/AOB neurons are less sensitive to stress caused by MS than preexisting ones. LAY SUMMARY Our results showed that single maternal separation (SMS) is a stressful event that in age-dependent manner stimulates cellular activity in the main and accessory olfactory bulb (AOB) - the structures dedicated to odor information processing. The low level of Fos expression in newborn neurons of the main and accessory bulb indicates that postnatally generated cells are less sensitive to neonatal stress than preexisting neurons.

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.

Disrupted migration and proliferation of neuroblasts after postnatal administration of angiogenesis inhibitor.

In adult rodents, neuroblasts originating from the subventricular zone migrate tangentially through the rostral migratory stream (RMS) toward the olfactory bulb where they differentiate into interneurons. Neuroblasts in the RMS migrate in chains for a long distance along specifically arranged blood vessels which promote their migration. Although blood vessels in the neurogenic region of the forebrain are present early in development, their rearrangement into this specific pattern takes place during the first postnatal weeks. Here we examined the relevance of this rearrangement to the migration-guiding "scaffold" for the neurogenic processes in the RMS such as cell migration and proliferation. To disturb the reorganization of blood vessels, endostatin - an inhibitor of angiogenesis, was administered systemically to rat pups during the first postnatal week. Ten days or three months later, the arrangement of blood vessels, migration and proliferation of cells in the RMS were assessed. As we expected, the inhibition of angiogenesis disrupted rearrangement of blood vessels in the RMS. The rearrangement's failure resulted in a strong disruption of the mode and direction of neuroblast migration. Chain migration failed and neuroblasts migrated out of the RMS. The inhibition caused a slight increase in the number of proliferating cells in the RMS. The consequences were more obvious ten days after the inhibition of angiogenesis, although they persisted partly into adulthood. Altogether, here we show that the process of rearrangement of blood vessels in the RMS during the early postal period is crucial to ensure the regular course of postnatal neurogenesis.

Analysis of Fos expression in the rat olfactory neurogenic region following single exposure to maternal separation during different neonatal stages.

Accumulating evidence confirms that the exposure of neonatal rats to maternal separation can significantly alter individual processes of postnatal neurogenesis in the olfactory neurogenic region - the subventricular zone (SVZ) and the rostral migratory stream (RMS). To establish the stressful influence of MS on postnatal neurogenesis we have investigated whether altered olfactory environment caused by short-term MS induces expression of Fos protein in the SVZ/RMS and in the olfactory cortical area - anterior olfactory nucleus (AON) of neonatal rats. Pups were separated from mothers for 2 hours at the postnatal days 7, 14 and 21. Immunohistochemically labeled Fos protein was assessed. Our results revealed that single exposure to MS is a stressful event that selectively and in age-dependent manner stimulates cellular activity in the SVZ and AON. A few Fos+ cells were found in the SVZ of P21 control animals and MS significantly increased their number. This suggests that some SVZ cells are included in the circuitry, which is activated by MS and that these cells have complete equipment for the Fos signal transduction. MS significantly increased the number of Fos+ cells in the AON in all age stages examined suggesting that its effect is mediated by olfaction.

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.

Repeated Baclofen treatment ameliorates motor dysfunction, suppresses reflex activity and decreases the expression of signaling proteins in reticular nuclei and lumbar motoneurons after spinal trauma in rats.

The interruption of supraspinal input to the spinal cord leads to motor dysfunction and the development of spasticity. Clinical studies have shown that Baclofen (a GABAB agonist), while effective in modulating spasticity is associated with side-effects and the development of tolerance. The aim of the present study was to assess if discontinued Baclofen treatment and its repeated application leads antispasticity effects, and whether such changes affect neuronal nitric oxide synthase (nNOS) in the brainstem, nNOS and parvalbumin (PV) in lumbar α-motoneurons and glial fibrillary acidic protein in the ventral horn of the spinal cord. Adult male Wistar rats were exposed to Th9 spinal cord transection. Baclofen (30mg/b.w.) diluted in drinking water, was administered for 6 days, starting at week 1 after injury and then repeated till week 4 after injury. The behavior of the animals was tested (tail-flick test, BBB locomotor score) from 1 to 8 weeks. Our results clearly indicate the role of nitric oxide, produced by nNOS in the initiation and the maintenance of spasticity states 1, 6 and 8 weeks after spinal trauma. A considerable decrease of nNOS staining after Baclofen treatment correlates with improvement of motor dysfunction. The findings also show that parvalbumin and astrocytes participate in the regulation of ion concentrations in the sub-acute phase after the injury.