Use of serotonin immunocytochemistry as a marker of injury severity after experimental spinal trauma in rats

doi:10.1016/0006-8993(88)91548-X

Brain Research

Volume 450, Issues 1–2, 31 May 1988, Pages 94-100

https://doi.org/10.1016/0006-8993(88)91548-X Get rights and content

Abstract

In experimental models of spinal cord trauma there is often a relatively poor correlation between light microscopic histological changes and motor recovery. Previously it was shown that spinal cord levels of immunoreactive TRH and substance P, by radioimmunoassay, are significantly reduced caudal to the injury site. Since much of the substance P and TRH in the spinal cord derives from cells within the ventral medulla, many of which also contain serotonin, we examined changes in serotonin immunoreactivity within the spinal cord caudal to the injury site in rats subjected to varying degrees of impact trauma to the thoracic cord. Reductions in immunocytochemical staining of serotonin in ventral gray matter of the lumbar region at two weeks after trauma were significantly correlated with the degree of injury severity as reflected by motor impairment. Changes in the region of the central canal, but not dorsal horn, were also correlated with injury severity. These findings indicate that serotonin immunocytochemical analysis may permit better correlation between anatomical and functional outcome after spinal cord injury than generally utilized light microscopic methods.

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Gsx1 promotes locomotor functional recovery after spinal cord injury
2021, Molecular Therapy
Citation Excerpt :
Neurotransmission of serotonin (5-HT) in the spinal cord is required for modulating sensory, motor, and autonomic functions, and positive 5-HT staining is associated with neuronal activity.45 After SCI, 5-HT-positive axons caudal to the injury site degenerate, while rostral to the injury site they sprout.46,47 Thus, we examined the 5-HT immunoactivity in spinal cord tissues isolated at 35 DPI.
Promoting residential cells, particularly endogenous neural stem and progenitor cells (NSPCs), for tissue regeneration represents a potential strategy for the treatment of spinal cord injury (SCI). However, adult NSPCs differentiate mainly into glial cells and contribute to glial scar formation at the site of injury. Gsx1 is known to regulate the generation of excitatory and inhibitory interneurons during embryonic development of the spinal cord. In this study, we show that lentivirus-mediated expression of Gsx1 increases the number of NSPCs in a mouse model of lateral hemisection SCI during the acute stage. Subsequently, Gsx1 expression increases the generation of glutamatergic and cholinergic interneurons and decreases the generation of GABAergic interneurons in the chronic stage of SCI. Importantly, Gsx1 reduces reactive astrogliosis and glial scar formation, promotes serotonin (5-HT) neuronal activity, and improves the locomotor function of the injured mice. Moreover, RNA sequencing (RNA-seq) analysis reveals that Gsx1-induced transcriptome regulation correlates with NSPC signaling, NSPC activation, neuronal differentiation, and inhibition of astrogliosis and scar formation. Collectively, our study provides molecular insights for Gsx1-mediated functional recovery and identifies the potential of Gsx1 gene therapy for injuries in the spinal cord and possibly other parts of the central nervous system.
Serotonergic mechanisms in spinal cord injury
2019, Experimental Neurology
Citation Excerpt :
Importantly, the response of 5-HT axons to SCI differs according to their location with respect to the injury site. Caudal to the lesion site, 5-HT axons degenerate and the degree of axonal degeneration positively correlates with lesion severity (Faden et al., 1988). In the rat spinal cord, 5-HT immunoreactivity is significantly reduced as compared to normal condition (Fig. 3A) immediately caudal to the lesion, but it is less affected further caudally 2 weeks after moderate thoracic (T9/T10) contusion [10 g dropped from 25 mm height] (Hayashi et al., 2010; Holmes et al., 2005) (Fig. 3B).
Spinal cord injury (SCI) is a tragic event causing irreversible losses of sensory, motor, and autonomic functions, that may also be associated with chronic neuropathic pain. Serotonin (5-HT) neurotransmission in the spinal cord is critical for modulating sensory, motor, and autonomic functions. Following SCI, 5-HT axons caudal to the lesion site degenerate, and the degree of axonal degeneration positively correlates with lesion severity. Rostral to the lesion, 5-HT axons sprout, irrespective of the severity of the injury. Unlike callosal fibers and cholinergic projections, 5-HT axons are more resistant to an inhibitory milieu and undergo active sprouting and regeneration after central nervous system (CNS) traumatism. Numerous studies suggest that a chronic increase in serotonergic neurotransmission promotes 5-HT axon sprouting in the intact CNS. Moreover, recent studies in invertebrates suggest that 5-HT has a pro-regenerative role in injured axons. Here we present a brief description of 5-HT discovery, 5-HT innervation of the CNS, and physiological functions of 5-HT in the spinal cord, including its role in controlling bladder function. We then present a comprehensive overview of changes in serotonergic axons after CNS damage, and discuss their plasticity upon altered 5-HT neurotransmitter levels. Subsequently, we provide an in-depth review of therapeutic approaches targeting 5-HT neurotransmission, as well as other pre-clinical strategies to promote an increase in re-growth of 5-HT axons, and their functional consequences in SCI animal models. Finally, we highlight recent findings signifying the direct role of 5-HT in axon regeneration and suggest strategies to further promote robust long-distance re-growth of 5-HT axons across the lesion site and eventually achieve functional recovery following SCI.
Transplantation of neural progenitor cells in chronic spinal cord injury
2016, Neuroscience
Citation Excerpt :
The raphe magnus and raphe pallidus are among brainstem nuclei with identified connections to the bladder and EUS pathway (Marson, 1997; Vizzard et al., 1997). Previous studies have established that serotonin immunoreactivity distal to the injury site is a good indicator of both SCI severity and the degree of recovery of somatic sensorimotor function (Faden et al., 1988; Wrathall et al., 1994; Teng and Wrathall, 1997; Pikov and Wrathall, 2001). Measurement of serotonin immunoreactivity (5-HT) suggests that recovery of detrusor–EUS coordination is associated with sparing of supraspinal projections to areas in the lumbosacral spinal cord that control bladder and EUS function (Pikov and Wrathall, 2001).
Previous studies demonstrated that neural progenitor cells (NPCs) transplanted into a subacute contusion injury improve motor, sensory, and bladder function. In this study we tested whether transplanted NPCs can also improve functional recovery after chronic spinal cord injury (SCI) alone or in combination with the reduction of glial scar and neurotrophic support. Adult rats received a T10 moderate contusion. Thirteen weeks after the injury they were divided into four groups and received either: 1. Medium (control), 2. NPC transplants, 3. NPC + lentivirus vector expressing chondroitinase, or 4. NPC + lentivirus vectors expressing chondroitinase and neurotrophic factors. During the 8 weeks post-transplantation the animals were tested for functional recovery and eventually analyzed by anatomical and immunohistochemical assays. The behavioral tests for motor and sensory function were performed before and after injury, and weekly after transplantation, with some animals also tested for bladder function at the end of the experiment. Transplant survival in the chronic injury model was variable and showed NPCs at the injury site in 60% of the animals in all transplantation groups. The NPC transplants comprised less than 40% of the injury site, without significant anatomical or histological differences among the groups. All groups also showed similar patterns of functional deficits and recovery in the 12 weeks after injury and in the 8 weeks after transplantation using the Basso, Beattie, and Bresnahan rating score, the grid test, and the Von Frey test for mechanical allodynia. A notable exception was group 4 (NPC together with chondroitinase and neurotrophins), which showed a significant improvement in bladder function. This study underscores the therapeutic challenges facing transplantation strategies in a chronic SCI in which even the inclusion of treatments designed to reduce scarring and increase neurotrophic support produce only modest functional improvements. Further studies will have to identify the combination of acute and chronic interventions that will augment the survival and efficacy of neural cell transplants.
Cograft of neural stem cells and schwann cells overexpressing TrkC and neurotrophin-3 respectively after rat spinal cord transection
2011, Biomaterials
Citation Excerpt :
Lastly, we did not detect Hoechst 33342 signal in cells adjacent to the lesion epicenter that had features of host neurons. Due to the fact that there was no indication of CST or RST regeneration based on neurofilament ICC (data not shown), we decided to focus on regional sprouting of serotonergic (5-HT) fibers from primary descending pathways of motosensory modulation in the mammalian spinal cord with known benefits to locomotion recovery [42–47]. Anti-5-HT ICC depicted that some serotonergic neurites were present inside the lesion site.
Effectively bridging the lesion gap is still an unmet demand for spinal cord repair. In the present study, we tested our hypothesis if cograft of Schwann cells (SCs) and neural stem cells (NSCs) with genetically enhanced expression of neurotrophin-3 (NT-3) and its high affinity receptor TrkC, respectively, could strengthen neural repair through increased NSC survival and neuronal differentiation at the epicenter after complete T10 spinal cord transection in adult rats. Transplantation of NT-3-SCs + TrkC-NSCs in Gelfoam (1 × 10⁶/implant/rat; n = 10) into the lesion gap immediately following injury results in significantly improved relay of the cortical motor evoked potential (CMEP) and cortical somatosensory evoked potential (CSEP) as well as ameliorated hindlimb deficits, relative to controls (treated with LacZ-SCs + LacZ-NSCs, NT-3-SCs + NSCs, NSCs alone, or lesion only; n = 10/group). Further analyses demonstrate that NT-3-SCs + TrkC-NSCs cografting augments levels of neuronal differentiation of NSCs, synaptogenesis (including inhibitory/type II-like synapses) and myelin formation of SCs, in addition to neuroprotection and outgrowth of serotonergic fibers in the lesioned spinal cord. Compared with controls, the treated spinal cords also show elevated expression of laminin, a pro-neurogenic factor, and decreased presence of chondroitin sulfate proteoglycans, major inhibitors of axonal growth and neuroplasticity. Together, our data suggests that coimplantation of neurologically compatible cells with compensatorily overexpressed therapeutic genes may constitute a valuable approach to study, and/or develop therapies for spinal cord injury (SCI).
The time course of serotonin 2A receptor expression after spinal transection of rats: An immunohistochemical study
2011, Neuroscience
Citation Excerpt :
Thus, the upregulation of 5-HT2AR in the lateral intermediate zone in the L1-2 segments is likely to be related with recovery of locomotion following a lesion since these segments contain critical elements of the locomotor central pattern generator (reviewed by Guertin, 2009) and 5-HT2AR have been shown to be critical for locomotor network activation (Ung et al., 2008; Halberstadt et al., 2009). The changes in 5-HT expression after spinalization in the present study are in agreement with previous biochemical and immunohistochemical studies (Anden et al., 1964; Clineschmidt et al., 1971; Magnusson, 1973; Hadjiconstantinou et al., 1984; Faden et al., 1988). Although intraspinal 5-HT neurons were reported to exist in the spinal cord even after spinal transection (Newton and Hamill, 1988; Kong et al., 2010), we did not detect any 5-HT fibers in the ventral horn motoneuron region in the analyzed sections in 21 days and 28 days spinalized animals.
Hyperexcitability of motoneurons is one of the key mechanism that has been demonstrated to underlie the pathogenesis of spasticity after spinal injury. Serotonin (5-HT) denervation supersensitivity is one of the mechanisms underlying this increased motoneuron excitability. In this study, to examine whether the supersensitivity is caused by 5-HT receptor upregulation we investigated changes in levels of 5-HT2A receptor immunoreactivity (5-HT2AR-IR) following a spinal transection in the sacral spinal cord of rats at seven different time points post injury: 2, 8, 16 h, and 1, 2, 7 and 28 days, respectively. 5-HT2AR-IR density was analyzed in motoneurons (regions containing their somata and dendrites) in the spinal segments below the lesion. The results showed no significant changes in 5-HT2AR-IR in the motoneurons up to 16 h following the transection. After 1-day, however the levels of 5-HT2AR-IR increased in the motoneurons and their dendrites, with the density level being 3.4-fold higher in spinalized rats than in sham-operated rats. The upregulation increased progressively until a maximal level was reached at 28 days post-injury. We also investigated 5-HT and 5-HT transporter expressions at five different post injury time points: 1, 2, 7, 21 and 60 days and they showed concurrent down-regulation changes after 2 days. These results suggest that the upregulation of 5-HT2ARs may at least partly underlie the development of 5-HT denervation supersensitivity in spinal motoneurons following spinal injury and thereby implicates their involvement in the pathogenesis of the subsequent development of spasticity.
Implantation of adult bone marrow-derived mesenchymal stem cells transfected with the neurotrophin-3 gene and pretreated with retinoic acid in completely transected spinal cord
2010, Brain Research
Implantation of marrow-derived mesenchymal stem cells (MSCs) is the most promising therapeutic strategy for the treatment of spinal cord injury (SCI), especially because of their potential for clinical application, such as the avoidance of immunologic rejection, their strong secretory properties, and their plasticity for developing into neural cells. However, the recovery from SCI after MSC implantation is minimal due to their limited capacity for the reduction of cystic cavitation, for the axonal regeneration and their uncertain neural plasticity in the spinal cord. We previously pretreated MSCs with all-trans retinoic acid (RA) in vitro. Then we genetically modified them to overexpress neurotrophin-3 (NT-3) via a recombinant adenoviral vector (Adv). This combined treatment not only permitted more neuronal differentiation of MSCs, but stimulated more NT-3 secretion prior to grafting, according to our previous and present results. When these cells were implanted into the transected spinal cord of rats, the animals had some improvement (both functionally and structurally), including the recovery of hindlimb locomotor function, shown by the highest Basso, Beattie, and Bresnahan (BBB) scores, as well as dramatically reduced cavity volume, clear axonal regeneration and more neuronal survival. In contrast, simple MSC implantation is not a very effective therapy for spinal transection. However, the neuronal differentiation of MSCs after treatment with a combination of Adv-mediated NT-3 gene transfer and RA was only mildly improved in vivo.

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Research reportUse of serotonin immunocytochemistry as a marker of injury severity after experimental spinal trauma in rats

Abstract

Neuroscience

Exp. Neurol.

J. Neurol. Sci.

Prog. Neurobiol.

Trends Neurosci.

Neuropeptides

Neuropeptides

Brain Research

Exp. Neurol.

Peptides

Brain Research

Neuroscience

Brain Research

Research report
Use of serotonin immunocytochemistry as a marker of injury severity after experimental spinal trauma in rats