promotion-survival-differentiation-neural-stem-cells-with-fibrin

Promotion of Survival and Differentiation of Neural Stem Cells with Fibrin and Growth Factor Cocktails after Severe Spinal Cord Injury

Neural stem cells (NSCs) can self-renew and differentiate into neurons and glia. Transplanted NSCs can replace lost neurons and glia after spinal cord ...

University of California, San Diego

We show that it is feasible to monitor the synchronized population spikes of the thalamocortical axonal terminals and cortical neurons outside the brain using high-resolution magnetoencephalography (MEG). Electrical stimulation of the snout elicited somatic-evoked magnetic fields (SEFs) above the primary somatosensory cortex (SI) of the piglet. The SEFs contained high-frequency oscillations (HFOs) around 600 Hz similar in many respects to the noninvasively measured HFOs from humans with MEG and electroencephalography (EEG). These HFOs were highly correlated with those in simultaneously measured intracortical somatic-evoked potentials (SEPs) in the snout projection area in SI. Both HFOs in SEFs and SEPs consisted of an initial component insensitive to cortically injected kynurenic acid (Kyna, 20 mM), a nonspecific antagonist of glutamatergic receptors, and a subsequent Kyna-sensitive component. The former was localized in cortical layer IV, indicating that it was due to spikes produced by the specific thalamocortical axonal terminals, whereas the latter was initially localized in layer IV and subsequently in the superficial and deeper layers. These results suggest that it may be possible to study properties of the thalamocortical and cortical spike activities in humans with MEG.

Synchronized spikes of thalamocortical axonal terminals and cortical neurons are detectable outside the pig brain with MEG.

The primary somatosensory cortex of various species including man, monkey, pig and rat is capable of producing high-frequency signals in the 600 Hz range and above with very little latency jitter. We have recently observed such cortical signals for the trigeminal system of the swine. This study determined the projection of the maxillary nerve innervating the snout to the sensory trigeminal nuclear complex in the brain stem and stability of outputs of each nucleus receiving the projection. The snout stimulation activated large-caliber Abeta fibers in the trigeminal nerve with a mean velocity of 64.4+/-2.7 m/s (mean+/-1 S.E.M., six animals) comparable in velocity to the tooth pulp Abeta fibers (57.9+/-3.4 m/s) obtained from the same animals. These afferents activated the main sensory nucleus, and subnuclei oralis, interpolaris and caudalis of the spinal nucleus, as judged by evoked field potential maps superimposed on the histological maps of the trigeminal nuclei from the same animals. Inputs from these fast afferents arrived at all the four trigeminal nuclei almost simultaneously within a span of 0.7+/-0.2 ms (mean+/-1 S.D., seven animals). Evoked high-frequency signals were reproducible with a latency jitter of less than 0.2 ms during the first 4 ms of postsynaptic activity for each of main sensory and spinal nuclei. These results indicate that the snout stimulation activates fast-conducting peripheral afferents which project to all the sensory trigeminal nuclei and produces highly reproducible initial responses nearly simultaneously across the multiple trigeminal nuclei. These outputs from the trigeminal nuclei may play an important role in triggering the stable high-frequency signals in the cortex.

Stable synchronized high-frequency signals from the main sensory and spinal nuclei of the pig activated by Abeta fibers of the maxillary nerve innervating the snout.

The absence of cortical responses to external stimuli is a dubious clinical sign during the first 1-2 days of brain injury. We previously showed that the amplitude of the somatic evoked potential (SEP) in the swine is diminished at the infarct site and perihematomal surround within the first 6 h of collagenase-induced intracerebral hemorrhage (ICH). We now report that this depressed SEP persists during the subchronic (48 h) period of ICH in the swine not only within the injured primary somatosensory (SI) cortex, but also in the contralateral homotopic SI cortex. This impairment of sensory responsiveness was accompanied by increases in various matrix metalloproteinases (MMPs) in different brain regions. By 24 h, a marked rise in MMP-9, an inflammatory marker, was detected in the white matter of the ipsilesional SI and secondary somatosensory cortex (SII), and in the contralesional SI gray matter, as compared to saline-injected controls. A subsequent increase in MMP-9 level was found in the ipsilesional SI and SII gray matter, and in the contralesional SI white matter by 48 h (P<0.05). By 7 days, significant levels of MMP-9 were detected only in the ipsilesional SI white and gray matter tissues. In contrast, the elevation of MMP-2, a marker of degeneration, was delayed until 7 days post-ICH in the ipsilesional SII gray matter. A significant rise in MMP-9 was also noted in CA1 of the ipsilesional and contralesional hemispheres during 1-2 days. Our MMP assay shows that the depressed cortical excitability seen in the contralateral SI cortex is a manifestation of the broad effect of a focal ICH that produces inflammatory and degenerative processes not only in the region adjacent to the focal ICH site, but also in remote regions that are functionally connected to the site of focal ICH.

Depressed cortical excitability and elevated matrix metalloproteinases in remote brain regions following intracerebral hemorrhage.

In humans, the somatic evoked potentials (SEPs) and magnetic fields (SEFs) elicited by peripheral nerve stimulation contain high-frequency oscillations (HFOs) around 600 Hz superimposed on the initial cortical response N20. Responses elicited by snout stimulation in the swine also contain similar HFOs during the rising phase of the porcine N20. This study examined the generators of the N20 and HFOs in the swine.

Origins of the somatic N20 and high-frequency oscillations evoked by trigeminal stimulation in the piglets.

Interpretation of Electroencephalography (EEG) signals from newborns is in some cases difficult because the fontanels and open sutures produce inhomogeneity in skull conductivity. We experimentally determined how EEG is influenced by a hole mimicking the anterior fontanel since distortion of EEG signals is important in neurological examinations during the perinatal period.

Evaluation of the distortion of EEG signals caused by a hole in the skull mimicking the fontanel in the skull of human neonates.

We studied effects of a temporary bilateral ligation of the internal carotid arteries on the subcortical and cortical structures of the somatosensory system by examining the thalamic input and postsynaptic cortical responses contained in the somatic evoked potentials (SEPs) recorded from the primary somatosensory cortex (SI) of the juvenile piglets in vivo. We predicted that the ligation should differentially affect these structures due to differences in blood supply.

Effects of temporary bilateral ligation of the internal carotid arteries on the low- and high-frequency somatic evoked potentials in the swine.

Substance P (SP) is thought to play a cardinal role in emesis via the activation of central tachykinin NK1 receptors during the delayed phase of vomiting produced by chemotherapeutics. Although the existing supportive evidence is significant, due to lack of an appropriate animal model, the evidence is indirect. As yet, no study has confirmed that emesis produced by SP or a selective NK1 receptor agonist is sensitive to brain penetrating antagonists of either NK1, NK2, or NK3 receptors. The goals of this investigation were to demonstrate: 1) whether intraperitoneal (i.p.) administration of either SP, a brain penetrating (GR73632) or non-penetrating (e.g. SarMet-SP) NK1 receptor agonist, an NK2 receptor agonist (GR64349), or an NK3 receptor agonist (Pro7-NKB), would induce vomiting and/or scratching in the least shrew (Cryptotis parva) in a dose-dependent manner; and whether these effects are sensitive to the above selective receptor antagonists; 2) whether an exogenous emetic dose of SP (50 mg/kg, i.p.) can penetrate into the shrew brain stem and frontal cortex; 3) whether GR73632 (2.5 mg/kg, i.p.)-induced activation of NK1 receptors increases Fos-measured neuronal activity in the neurons of both brain stem emetic nuclei and the enteric nervous system of the gut; and 4) whether selective ablation of peripheral NK1 receptors can affect emesis produced by GR73632. The results clearly demonstrated that while SP produced vomiting only, GR73632 caused both emesis and scratching behavior dose-dependently in shrews, and these effects were sensitive to NK1-, but not NK2- or NK3-receptor antagonists. Neither the selective, non-penetrating NK1 receptor agonists, nor the selective NK2- or NK3-receptor agonists, caused a significant dose-dependent behavioral effect. An emetic dose of SP selectively and rapidly penetrated the brain stem but not the frontal cortex. Systemic GR73632 increased Fos expression in the enteric nerve plexi, the medial subnucleus of nucleus tractus solitarius, and the dorsal motor nucleus of the vagus, but not the area postrema. Ablation of peripheral NK1 receptors attenuated the ability of GR73632 to induce a maximal frequency of emesis and shifted its percent animals vomiting dose-response curve to the right. The NK1-ablated shrews exhibited scratching behavior after systemic GR73632-injection. These results, for the first time, affirm a cardinal role for central NK1 receptors in SP-induced vomiting, and a facilitatory role for gastrointestinal NK1 receptors. In addition, these data support the validation of the least shrew as a specific and rapid behavioral animal model to screen concomitantly both the CNS penetration and the antiemetic potential of tachykinin NK1 receptor antagonists.

Utilization of the least shrew as a rapid and selective screening model for the antiemetic potential and brain penetration of substance P and NK1 receptor antagonists.

5-HT3 receptor antagonists (e.g. tropisetron) combined with dexamethasone are effective for the acute phase of cisplatin (CIS)-induced emesis. This study determined the possible additive or synergistic antiemetic efficacy of Delta9-THC when combined with tropisetron or dexamethasone (DEX). Delta9-THC (0-10 mg/kg i.p.) was injected in combination with tropisetron (0-5 mg/kg i.p.) or dexamethasone (0-20 mg/kg i.p.) prior to CIS (20 mg/kg i.p.) in the least shrew, and the induced emesis was recorded for 60 min. CIS-induced vomiting was dose-dependently and significantly attenuated by individual administration of Delta9-THC (59-97% reductions) and tropisetron (79-100% attenuation), but not dexamethasone (26-40%), although a trend (p<0.1) towards reduced vomiting frequency following DEX was noted. Low doses of Delta9-THC (0.25 or 0.5 mg/kg) when combined with low doses of tropisetron (0.025, 0.1, or 0.25 mg/kg) were more efficacious in reducing emesis frequency than when given individually, but Delta9-THC had no antiemetic interactions with DEX. However, no tested combination provided a significantly greater effect on the number of animals vomiting than their individually-administered counterparts. The modest interaction of Delta9-THC with tropisetron suggests they activate overlapping antiemetic mechanisms, while the lack of interaction with dexamethasone needs further clarification.

The antiemetic interaction of Delta9-tetrahydrocannabinol when combined with tropisetron or dexamethasone in the least shrew.

Many chemotherapeutic agents activate multiple signaling systems, including potentially emetogenic arachidonic acid metabolites. Of these messengers, the emetic role of the leukotriene family has been neglected. The aims of this study were to test the emetic potential of key leukotrienes (LTA(4), LTB(4), LTF(4), and the cysteinyl leukotrienes LTC(4), LTD(4) and LTE(4)), and to investigate whether the leukotriene CysLT(1) receptor antagonist pranlukast or mixed leukotriene CysLT(1/2) receptor antagonist Bay u9773 can prevent the LTC(4)-induced emesis. Least shrews were injected with varying doses of one of the six tested leukotrienes and vomiting parameters were measured for 30min. LTC(4) and LTD(4) were most efficacious, and significantly increased both the frequency and percentage of animals vomiting at doses from 0.1 and 0.05mg/kg, respectively. The other tested leukotrienes were either weakly emetic or ineffective at doses up to 4mg/kg. The relative emetogenic activities of the cysteinyl leukotrienes (LTC(4)=LTD(4)>LTE(4)) suggest that leukotriene CysLT(2) receptors have a key role in emesis. However, pranlukast dose-dependently, and at 10mg/kg completely, blocked LTC(4)-induced vomiting, implicating a leukotriene CysLT(1) receptor-mediated emetic effect. Bay u9773 dose-dependently reduced the percentage of animals vomiting, but did not significantly reduce vomiting frequency. Fos immunoreactivity, measured subsequent to LTC(4)-induced vomiting to define its putative anatomical substrates, was significantly increased in the enteric nervous system and medullary dorsal vagal complex following LTC(4) (P<0.05) versus vehicle injections. This study is the first to show that some leukotrienes induce emesis, possibly involving both central and peripheral leukotriene CysLT(1) and/or leukotriene CysLT(2) receptors.

Pranlukast prevents cysteinyl leukotriene-induced emesis in the least shrew (Cryptotis parva).

We subjected rats to either partial midcervical or complete upper thoracic spinal cord transections and examined whether combinatorial treatments support motor axonal regeneration into and beyond the lesion. Subjects received cAMP injections into brainstem reticular motor neurons to stimulate their endogenous growth state, bone marrow stromal cell grafts in lesion sites to provide permissive matrices for axonal growth, and brain-derived neurotrophic factor gradients beyond the lesion to stimulate distal growth of motor axons. Findings were compared with several control groups. Combinatorial treatment generated motor axon regeneration beyond both C5 hemisection and T3 complete transection sites. Yet despite formation of synapses with neurons below the lesion, motor outcomes worsened after partial cervical lesions and spasticity worsened after complete transection. These findings highlight the complexity of spinal cord repair and the need for additional control and shaping of axonal regeneration.

Motor axonal regeneration after partial and complete spinal cord transection.

Neural stem cells (NSCs) expressing GFP were embedded into fibrin matrices containing growth factor cocktails and grafted to sites of severe spinal cord injury. Grafted cells differentiated into multiple cellular phenotypes, including neurons, which extended large numbers of axons over remarkable distances. Extending axons formed abundant synapses with host cells. Axonal growth was partially dependent on mammalian target of rapamycin (mTOR), but not Nogo signaling. Grafted neurons supported formation of electrophysiological relays across sites of complete spinal transection, resulting in functional recovery. Two human stem cell lines (566RSC and HUES7) embedded in growth-factor-containing fibrin exhibited similar growth, and 566RSC cells supported functional recovery. Thus, properties intrinsic to early-stage neurons can overcome the inhibitory milieu of the injured adult spinal cord to mount remarkable axonal growth, resulting in formation of new relay circuits that significantly improve function. These therapeutic properties extend across stem cell sources and species.

Article	Year
Synchronized spikes of thalamocortical axonal terminals and cortical neurons are detectable outside the pig brain with MEG. Journal of neurophysiology\| PubMed ID: 11784777	2002
Stable synchronized high-frequency signals from the main sensory and spinal nuclei of the pig activated by Abeta fibers of the maxillary nerve innervating the snout. Brain research\| PubMed ID: 12480152	2003
Depressed cortical excitability and elevated matrix metalloproteinases in remote brain regions following intracerebral hemorrhage. Brain research\| PubMed ID: 15488484	2004
Origins of the somatic N20 and high-frequency oscillations evoked by trigeminal stimulation in the piglets. Clinical neurophysiology : official journal of the International Federation of Clinical Neurophysiology\| PubMed ID: 15792892	2005
Evaluation of the distortion of EEG signals caused by a hole in the skull mimicking the fontanel in the skull of human neonates. Clinical neurophysiology : official journal of the International Federation of Clinical Neurophysiology\| PubMed ID: 15826855	2005
Effects of temporary bilateral ligation of the internal carotid arteries on the low- and high-frequency somatic evoked potentials in the swine. Clinical neurophysiology : official journal of the International Federation of Clinical Neurophysiology\| PubMed ID: 16125462	2005
Utilization of the least shrew as a rapid and selective screening model for the antiemetic potential and brain penetration of substance P and NK1 receptor antagonists. Brain research\| PubMed ID: 18471804	2008
The antiemetic interaction of Delta9-tetrahydrocannabinol when combined with tropisetron or dexamethasone in the least shrew. Pharmacology, biochemistry, and behavior\| PubMed ID: 18727934	2009
Pranlukast prevents cysteinyl leukotriene-induced emesis in the least shrew (Cryptotis parva). European journal of pharmacology\| PubMed ID: 19941848	2010
Motor axonal regeneration after partial and complete spinal cord transection. The Journal of neuroscience : the official journal of the Society for Neuroscience\| PubMed ID: 22699902	2012
Long-distance growth and connectivity of neural stem cells after severe spinal cord injury. Cell\| PubMed ID: 22980985	2012
Neural stem cell dissemination after grafting to CNS injury sites. Cell\| PubMed ID: 24485445	2014

Yaozhi Wang

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