Triggering of Autophagy by Baicalein in Response to Apoptosis after Spinal Cord Injury: Possible Involvement of the PI3K Activation.

High level apoptosis induced by spinal cord injury (SCI) evokes serious damage because of the loss and dysfunction of motor neurons. Our previous studies showed that inhibition of autophagy evokes the activation of apoptosis. Interestingly, Baicalein, a medicine with anti-apoptosis activity that is derived from the roots of herb Scutellaria baicalensis, largely induces autophagy by activating phosphatidylinositol 3-kinase (PI3K). In this study, we investigated the effects of intraperitoneal injection of Baicalein on autophagy and apoptosis in SCI mice and evaluated the relationship between autophagy and apoptosis. We demonstrated that Baicalein promoted the functional recovery of motor neurons at 7 d after SCI. In addition, Baicalein enhanced neuronal autophagy and the autophagy-related factor PI3K, while inhibiting the p62 protein. Baicalein treatment decreased neuronal apoptosis at 7 d after SCI. Moreover, when inhibiting autophagy, apoptosis was upgraded by Baicalein treatment after injury. Thus, Baicalein attenuated SCI by inducing autophagy to reduce apoptosis in neurons potentially via activating PI3K.

Tail Nerve Electrical Stimulation and Electro-Acupuncture Can Protect Spinal Motor Neurons and Alleviate Muscle Atrophy after Spinal Cord Transection in Rats.

Spinal cord injury (SCI) often results in death of spinal neurons and atrophy of muscles which they govern. Thus, following SCI, reorganizing the lumbar spinal sensorimotor pathways is crucial to alleviate muscle atrophy. Tail nerve electrical stimulation (TANES) has been shown to activate the central pattern generator (CPG) and improve the locomotion recovery of spinal contused rats. Electroacupuncture (EA) is a traditional Chinese medical practice which has been proven to have a neural protective effect. Here, we examined the effects of TANES and EA on lumbar motor neurons and hindlimb muscle in spinal transected rats, respectively. From the third day postsurgery, rats in the TANES group were treated 5 times a week and those in the EA group were treated once every other day. Four weeks later, both TANES and EA showed a significant impact in promoting survival of lumbar motor neurons and expression of choline acetyltransferase (ChAT) and ameliorating atrophy of hindlimb muscle after SCI. Meanwhile, the expression of neurotrophin-3 (NT-3) in the same spinal cord segment was significantly increased. These findings suggest that TANES and EA can augment the expression of NT-3 in the lumbar spinal cord that appears to protect the motor neurons as well as alleviate muscle atrophy.

Light-induced rescue of breathing after spinal cord injury.

Paralysis is a major consequence of spinal cord injury (SCI). After cervical SCI, respiratory deficits can result through interruption of descending presynaptic inputs to respiratory motor neurons in the spinal cord. Expression of channelrhodopsin-2 (ChR2) and photostimulation in neurons affects neuronal excitability and produces action potentials without any kind of presynaptic inputs. We hypothesized that after transducing spinal neurons in and around the phrenic motor pool to express ChR2, photostimulation would restore respiratory motor function in cervical SCI adult animals. Here we show that light activation of ChR2-expressing animals was sufficient to bring about recovery of respiratory diaphragmatic motor activity. Furthermore, robust rhythmic activity persisted long after photostimulation had ceased. This recovery was accomplished through a form of respiratory plasticity and spinal adaptation which is NMDA receptor dependent. These data suggest a novel, minimally invasive therapeutic avenue to exercise denervated circuitry and/or restore motor function after SCI.

Immunohistochemical and hodological characterization of calbindin-D28k-containing neurons in the spinal cord of the turtle, Pseudemys scripta elegans.

Neurons and fibers containing the calcium-binding protein calbindin-D28k (CB) were studied by immunohistochemical techniques in the spinal cord of adult and juvenile turtles, Pseudemys scripta elegans. Abundant cell bodies and fibers immunoreactive for CB were widely and distinctly distributed throughout the spinal cord. Most neurons and fibers were labeled in the superficial dorsal horn, but numerous cells were also located in the intermediate gray and ventral horn. In the dorsal horn, most CB-containing cells were located in close relation to the synaptic fields formed by primary afferents, which were not labeled for CB. Double immunohistofluorescence demonstrated distinct cell populations in the dorsal horn labeled only for CB or nitric oxide synthase, whereas in the dorsal part of the ventral horn colocalization of nitric oxide synthase was found in about 6% of the CB-immunoreactive cells in this region. Choline acetyltransferase immunohistochemistry revealed that only about 2% of the neurons in the dorsal part of the ventral horn colocalized CB, whereas motoneurons were not CB-immunoreactive. The involvement of CB-containing neurons in ascending spinal projections to the thalamus, tegmentum, and reticular formation was demonstrated combining the retrograde transport of dextran amines and immunohistochemistry. Similar experiments demonstrated supraspinal projections from CB-containing cells mainly located in the reticular formation but also in the thalamus and the vestibular nucleus. The revealed organization of the neurons and fibers containing CB in the spinal cord of the turtle shares distribution and developmental features, colocalization with other neuronal markers, and connectivity with other tetrapods and, in particular with mammals.

SMN, the product of the spinal muscular atrophy-determining gene, is expressed widely but selectively in the developing human forebrain.

The expression pattern of the survival motor neuron (SMN) protein has been investigated immunohistochemically in the human fetal forebrain from 14 to 38 weeks of gestation. Mutations in the SMN gene cause spinal muscular atrophy (SMA), an autosomal recessive disease characterized by degeneration of lower motor neurons in the spinal cord leading to progressive muscle wasting. SMN is a multifunctional protein and has been implicated in diverse cytoplasmic and nuclear processes. The monoclonal murine SMN antibody used in this study recognized a major band at approximately 34 kDa. In spinal cord anterior horn motor neurons at 13 weeks of gestation, the soma, proximal neurites, and nucleus were immunostained. In the nucleus, SMN immunolabeling was observed at the nuclear membrane, at the nucleolus, and at dot-like structures in the nucleoplasm likely to be coiled bodies and gems. In the fetal forebrain, SMN was immunodetected as early as 14 weeks of gestation. From 14 to 24 weeks of gestation, intense immunostaining was observed in the basal nucleus of Meynert, a major source of cholinergic afferents to the cortex. Less intensely labeled cells at lower packing density were also observed in the thalamus, reticular and perireticular nucleus, globus pallidus, hippocampus, amygdala, and enthorinal cortex. Immunolabeled cells were still detectable at 38 gestational weeks, the latest time point investigated. These findings provide an anatomical basis for future investigations of SMN functions during brain development and for the neuropathological characterization of severe SMA cases.

Molecular mapping of developing dorsal horn-enriched genes by microarray and dorsal/ventral subtractive screening.

The dorsal horn of the spinal cord consists of distinct laminae that serve as a pivotal region for relaying a variety of somatosensory signals such as temperature, pain, and touch. The molecular mechanisms underlying the development of the dorsal horn are poorly understood. To define a molecular map of the dorsal horn circuit, we have profiled dorsal horn-enriched (DHE) gene expression in dorsal spinal cords on embryonic day 15.5 (E15.5) by genome-wide microarray and smart subtractive screening based on polymerase chain reaction (PCR). High-throughput in situ hybridization (ISH) was carried out to validate the expression of 379 genes in the developing dorsal spinal cord. A total of 113 DHE genes were identified, of which 59% show lamina-specific expression patterns. Most lamina-specific genes were expressed across at least two laminae, however. About 32% of all DHE genes are transcription factors, which represent the largest percentage of the group of all DHE functional classifications. Importantly, several individual lamina-specific transcription factors such c-Maf, Rora, and Satb1 are identified for the first time. Epistasis studies revealed several putative effectors of known DHE transcription factors such as Drg11, Tlx3(Rnx), and Lmx1b. These effector genes, including Grp, Trpc3, Pcp4, and Enc1, have been implicated in synaptic transmission, calcium homeostasis, and structural function and thus may have similar roles in the dorsal horn. The identification of a large number of DHE genes, especially those that are lamina specific, lays a foundation for future studies on the molecular machinery that controls the development of the dorsal horn and on functional differences of these distinct laminae in the dorsal spinal cord.

Enhancement of BDNF and activated-ERK immunoreactivity in spinal motor neurons after peripheral administration of BDNF.

Brain-derived neurotrophic factor (BDNF) shows neurotrophic effects on adult motor neurons when given systemically, But it is unknown whether systemically administered BDNF is transported to central cell bodies to affect them directly. Here we used immunohistochemistry to investigate the transport of peripherally injected BDNF to spinal motor neurons and the subsequent activation of a signaling pathway. We first injected BDNF into the flexor digitorum brevis (FDB) and analyzed the motor nucleus that projects to the FDB for BDNF immunoreactivity (BDNF-ir) and phosphorylated extracellular signal-regulated kinase (ERK) 1/2 immunoreactivity (pERK1/2-ir). Both immunoreactivities were observed in the motor neuron cell bodies. Next, BDNF was injected subcutaneously (s.c.) into rats with a unilaterally axotomized sciatic nerve. pERK1/2-ir was detected in motor neurons of the lesioned side. BDNF-ir and pERK1/2-ir were also observed on the unlesioned side when a high dose of BDNF was injected. Therefore, we examined BDNF-ir and pERK1/2-ir after injecting BDNF s.c. into normal rats. Both immunoreactivities were observed in motor nuclei on both sides. Finally, we examined pERK1/2-ir after a lower dose of BDNF was injected, which prevents the decrease in choline acetyl transferase that occurs in the motor neuron upon axotomy. Spinal motor nuclei contained a few cell bodies with pERK1/2-ir. These findings represent the first direct evidence that subcutaneously injected BDNF is transported to motor neurons and that it activates a signaling pathway in the spinal cord and exhibits neurotrophic effects in vivo.