Xi-Ying Jiao

Strong Expression of Interleukin-1 Receptor Type I in the Rat Carotid Body

One of the unsolved key questions in neuroimmunomodulation is how peripheral immune signals are transmitted to the brain. It has been reported that the vagus might play a role in this regard. The underlying mechanism for this immune system-to-brain communication route is related to the binding of cytokines, such as interleukin (IL)-1β originating from activated immune cells, to their receptors in glomus cells of the vagal paraganglia. The existence of IL-1 receptor type I (IL-1RI) in vagal paraganglia has been proved. On the basis of these studies, a hypothesis is raised that the carotid body, as the largest paraganglion, might play a similar role to that of its abdominal partner. In this study we examined the distribution of IL-1RI in the carotid body by immunohistochemistry (IHC) and Western blotting techniques. The IHC results showed that almost all glomus cells in the carotid body displayed strong IL-1RI immunoreactivity. The IL-1RI-immunoreactive products were localized in the cytoplasm, nucleus, and cell membrane of the glomus cells. The Western blotting results also confirmed the existence of IL-1RI in both membranous and cytoplasmic elements of the carotid body. These results imply that the carotid body not only serves as a chemoreceptor for modulation of cardiorespiratory performance, as traditionally recognized, but also acts as a cytokine chemorereceptor for sensing immune signals.

Death of axotomized retinal ganglion cells delayed after intraoptic nerve transplantation of olfactory ensheathing cells in adult rats.

Intraorbital transection of the optic nerve (ON) always induces ultimate apoptosis of retinal ganglion cells (RGCs) and consequently irreversible defects of vision function. It was demonstrated that transplanted olfactory ensheathing cells (OECs) in partially injured spinal cord have a distant in vivo neuroprotective effect on descending cortical and brain stem neurons. However, this study gave no answers to the question whether OECs can protect the central sensitive neurons with a closer axonal injury because different neurons respond variously to similar axonal injury and the distance between the neuronal soma and axonal injury site has a definite effect on the severity of neuronal response and apoptosis. In the present study, we investigated the effect of transplanted OECs on RGCs after intraorbital ON transection in adult rats. Green fluorescent protein (GFP)-OECs were injected into the ocular stumps of transected ON and a significantly higher number of surviving RGCs was found together with a consistent marked increase in the mRNA and protein levels of BDNF in the ON stump and retina in the OEC-treated group at 7 days, but not 2 and 14 days, time point when compared to the control group. Our findings suggest that OEC transplantation induces the expression of BDNF in the ocular ON stump and retina and delays the death of axotomized RGCs at a certain survival period.

EXTRAVASATION OF BLOOD-BORNE IMMUNOGLOBULIN G THROUGH BLOOD-BRAIN BARRIER DURING ADRENALINE-INDUCED TRANSIENT HYPERTENSION IN THE RAT

The effect of transient hypertension on blood-brain barrier (BBB) permeability, particularly on extravasation of immunoglobulin G (IgG), has not been fully understood. In the present experiment, we investigated- the time course of endogenous albumin and IgG extravasation through BBB and the localization of extravasated IgG in brain parenchyma during- adrenaline (AD)-induced transient hypertension in the rat by using Evans blue fluorescence, immunohistochemistry, and Western blot. The results showed that a bolus injection of AD (10 μg/kg) induced a transient elevation of arterial pressure lasting about 1 min. The endogenous albumin and IgG entered the brain parenchyma via BBB only when hyper--tension occurred. Electron microscopically, the IgG-like immunoreactivities were predominantly seen in the cytoplasm of endothelia of capillaries, pericytes, extracellular space of parenchyma, and the cytoplasm of glial cells. The results suggest that circulating IgG or antibodies might contact the structures of brain parenchyma through passage of BBB when its permeability is temporally changed by transient hypertension. This phenomenon implies a possible mechanism of pathogenesis for immune-mediated diseases in the brain.

Intraneuronal localization of Nogo-A in the rat

Nogo‐A is known to be a myelin‐associated protein with strong inhibitory effect on neurite outgrowth and has been considered one of the major factors that hinder fiber regeneration in the central nervous system. Recent studies have demonstrated widespread occurrence of nogo‐A mRNA and Nogo‐A protein in neurons. Our concurrent immunohistochemical study substantiated the widespread distribution of neuronal Nogo‐A. The present study was thus focused on its intraneuronal distribution in the central nervous system, using Western blotting, immunohistochemical, and immunogold electron microscopic techniques. Western blotting of the nucleus, cytoplasm, and membrane subcellular fractions of the cerebellum and spinal cord tissues demonstrated that all three fractions contained Nogo‐A. Nogo‐A immunoreactivity could be identified under confocal microscope in the nucleus, perikayon, and proximal dendrite and along the cell membrane. Under the electron microscope, the perikaryonal Nogo‐A immunogold particles were mainly distributed at polyribosomes and rough endoplasmic reticulum, suggesting its relationship with translation process. The immunogold particles could also be found beneath or on the plasma membrane. In the nucleus, the Nogo‐A immunogold particles were found to be localized at the chromatins of the nucleus, indicating its possible involvement in gene transcription. The presence of Nogo‐A in the nucleus was further supported by transfection of COS‐7L cells with nogo‐A. This study provides the first immunocytochemical evidence for intraneuronal distribution of Nogo‐A. Apparently, the significance of Nogo‐A in the central nervous system is far more complex than what has been envisioned. J. Comp. Neurol. 458:1–10, 2003.

Activity of p44/42 MAP kinase in the caudal subnucleus of trigeminal spinal nucleus is increased following perioral noxious stimulation in the mouse

The extracellular signal-regulated protein kinase-1 and -2 (ERK1 and ERK2), also referred to as the p44/42 mitogen-activated protein kinase (p44/42 MAP kinase), plays an essential role in neuronal signal transduction, but its function involved in nociceptive response has not been deeply studied yet. Here we report immunohistochemical evidence that p44/42 MAPK might be critical in nociceptive response. We found that after formalin was injected into the perioral skin of the upper lip of mice, the number of activated p44/42 MAPK-like immunoreactive neurons was significantly increased in the laminae I and II of the caudal subnucleus of the trigeminal spinal nucleus (Sp5C). The positive neurons and fibers were mostly concentrated in the middle portion of Sp5C dorsoventrally, where the afferent fibers innervating the skin of the upper lip are terminated. The reactive products were localized in perikarya, dendrites, nuclei, and diffusely in the neuropil. The present result suggests that p44/42 MAPK may be important in the transmission and modulation of noxious information in Sp5C.

GABAergic and glycinergic synapses onto neurokinin-1 receptor-immunoreactive neurons in the pre-Bötzinger complex of rats: light and electron microscopic studies

The pre‐Bötzinger complex (preBötC) in the ventrolateral medulla is thought to be the kernel for respiratory rhythm generation. Neurons in the preBötC contain intense neurokinin‐1 receptor (NK1R) immunoreactivity. Some of these neurons in the adult preBötC are presumed to be the pre‐inspiratory interneurons that are essential for generating respiratory rhythm in the neonate. Chloride‐mediated synaptic inhibition is critical for rhythmogenesis in the adult. The present study used immunofluorescence histochemistry and immunogold‐silver staining to determine the inhibitory synaptic relationship between glutamic acid decarboxylase (GAD)‐ or glycine transporter 2 (GlyT2)‐immunoreactive (ir) boutons and NK1R‐ir neurons in the preBötC of adult rats. Under the confocal microscope, we found that GAD‐ and GlyT2‐ir boutons were in close apposition to NK1R‐ir somas and dendrites in the preBötC. Under the electron microscope, GAD‐ and GlyT2‐ir terminals were in close apposition to NK1R‐ir somas and dendrites. Symmetric synapses were identified between GAD‐ or GlyT2‐ir terminals and NK1R‐ir neurons. A total of 51.6% GAD‐ir and 38.2% GlyT2‐ir terminals were found to contact or make synapses with NK1R‐ir profiles, respectively. GAD‐ and GlyT2‐ir terminals synapsed not only upon NK1R‐ir neurons but also upon NK1R immuno‐negative neurons. NK1R‐ir neurons received both symmetric (presumed inhibitory) and asymmetric (presumed excitatory) synapses. Thus, the present findings provide the morphological basis for inhibitory inputs to NK1R‐ir neurons in the preBötC, consistent with the suggestion that chloride‐mediated synaptic inhibition may contribute importantly to rhythm generation by controlling the membrane potential trajectory and resetting rhythmic bursting of the kernel neurons in the adult.

In vitro beneficial activation of microglial cells by mechanically-injured astrocytes enhances the synthesis and secretion of BDNF through p38MAPK.

It has long been promulgated that microglial cells serve beneficial roles in the central nervous system (CNS). The beneficial role of microglial cells is considered to be linked with microglial activation and consequent up-regulation of various trophic factors. However, what triggers microglial activation and consequent elevated level of trophic factors, especially brain-derived neurotrophic factor (BDNF), following traumatic CNS injury has become a crucial but elusive issue. Furthermore, an effort still remains in understanding of the cellular and molecular mechanisms underlying the endogenous neuroprotection of activated microglial cells. In this study, we demonstrated that mechanically-injured astrocyte conditioned medium (ACM) could provoke beneficial activation of microglial cells and thus promote the transcription, synthesis and release of BDNF in cultured microglial cells. The microglia-derived BDNF can exerted a demonstrable biological role in promoting neurite outgrowth and intimate terminal contacts of dorsal root ganglion (DRG) neurons co-cultured with microglial cells. Moreover, ACM induced remarkable p38MAPK phosphorylation in cultured microglial cells that preceded the burst of BDNF. Activating p38-MAPK by anisomycin resulted in salutary effects similar to those seen with ACM, whereas specific inhibition of the p38MAPK by SB203580 abrogated all the positive effects of ACM, including BDNF promotion and subsequent neurite outgrowth of DRG neurite outgrowth of DRG neurons and their intimate terminal contacts with microglial cells. Together, our results indicated that the neuroprotection of the microglial source is mainly caused by micro-environmental soluble molecules released from injured astrocytes, and ACM-induced BDNF production and release from microglial cells may be mediated through p38-MAPK signaling pathway. Therefore, these findings may lay a foundation to further investigations on the microglial beneficial activation role in the repair of traumatic CNS injury and neurodegenerative diseases.

Sonic hedgehog released from scratch-injured astrocytes is a key signal necessary but not sufficient for the astrocyte de-differentiation

Recent studies demonstrated that mature atrocytes have the capacity for de-differentiating into neural stem/progenitor cells (NSPCs) in vitro and in vivo. However, it is still unknown what signals endow astroglial cells with a de-differentiation potential. Furthermore, the signaling molecules and underlying mechanism that confer astrocytes with the competence of NSPC phenotypes have not been completely elucidated. Here, we found that sonic hedgehog (Shh) production in astrocytes following mechanical injury was significantly elevated, and that incubation of astrocyes with the injured astrocyte conditioned medium (ACM) causes astrocytes to gradually lose their immunophenotypical profiles, and acquire NSPC characteristics, as demonstrated by down-regulation of typical astrocytic markers (GFAP and S100) and up-regulation of markers that are generally expressed in NSCs, (nestin, Sox2, and CD133). ACM treated astrocytes exhibit self-renewal capacity and multipotency similar to NSPCs. Concomitantly, in addition to Ptc, there was a significant up-regulation of the Shh downstream signal components Gli2 and Cyclin D1 which are involved in cell proliferation, dramatic changes in cell morphology, and the disruption of cell-cycle G1 arrest. Conversely, the depletion of Shh by administration of its neutralizing antibody (Shh n-Ab) effectively inhibited the de-differentiation process. Strikingly, Shh alone had little effect on astrocyte de-differentiation to NSPCs. These data above suggest that Shh is a key instructive molecule while other molecules secreted from insulted astrocytes may synergistically promote the de-differentiation event.

Evidence for Heterogeneity of Astrocyte De-Differentiation in vitro: Astrocytes Transform into Intermediate Precursor Cells Following Induction of ACM from Scratch-Insulted Astrocytes

Our previous study definitely demonstrated that the mature astrocytes could undergo a de-differentiation process and further transform into pluripotential neural stem cells (NSCs), which might well arise from the effect of diffusible factors released from scratch-insulted astrocytes. However, these neurospheres passaged from one neurosphere-derived from de-differentiated astrocytes possessed a completely distinct characteristic in the differentiation behavior, namely heterogeneity of differentiation. The heterogeneity in cell differentiation has become a crucial but elusive issue. In this study, we show that purified astrocytes could de-differentiate into intermediate precursor cells (IPCs) with addition of scratch-insulted astrocyte-conditioned medium (ACM) to the culture, which can express NG2 and A2B5, the IPCs markers. Apart from the number of NG2+ and A2B5+ cells, the percentage of proliferative cells as labeled with BrdU progressively increased with prolonged culture period ranging from 1 to 10 days. Meanwhile, the protein level of A2B5 in cells also increased significantly. These results revealed that not all astrocytes could de-differentiate fully into NSCs directly when induced by ACM, rather they generated intermediate or more restricted precursor cells that might undergo progressive de-differentiation to generate NSCs.

Significance of fixation of the vertebral column for spinal cord injury experiments.

Study Design. Thoracic spinal cord transections were performed in adult rats. The animals were divided into two groups, with or without internal fixation of the involved vertebral column. Histologic and immunohistochemical studies were performed to compare the effect of internal fixation of the vertebral column. Objectives. To find out the aspects and extent of beneficial effects of vertebral column fixation for spinal cord repair. Summary of Background Data. Vertebral column fixation is a routine procedure in clinical spinal cord surgery. Paradoxically, most, if not all, animal spinal cord experiments seem to have ignored the importance of vertebral column fixation. During trunk movements, the vertebral column flexes to different directions, accompanied by bending of the spinal cord. Following spinal cord lesions, with frequent bending of the cord there will be repeated bleeding, inflammation, and other pathologic processes at the lesion site. Thus, the healing process will be hampered. The severity of the damages that will be brought about by bending of the cord is, to a certain degree, unpredictable. There will be rather big individual variations in injury and repair among the same type of experiments, rendering quantification and conclusion difficult. Methods. Adult Sprague-Dawley rats were used. The thoracic spinal cord was transected. Strong stainless steel wires were used for internal fixation of the vertebral column. The histology of the horizontal sections of the spinal cord segment, which included the lesion site, was examined at the 14th postoperative day. The volumes of the secondary degeneration and meningeal scar, the gap between the borders of the proximal and distal stumps of the transected spinal cord, the thickness of the meningeal scar, the astrocytic reaction, and the abundance of regenerating nerve fibers at the lesion site were compared between the vertebral column fixed and nonfixed groups. Whenever possible, the results were evaluated quantitatively. Results. In all these aspects, the internally fixed group was consistently far better than the unfixed group. The quantitative analyses were as follows (fixed/unfixed): 1)volume of secondary degeneration: 1.07 ± 0.20/1.81 ± 0.43 mm3 (P < 0.01); 2) volume of meningeal scar: 2.38 ± 0.55/4.34 ± 1.40 mm3 (P < 0.05); 3) distance between cord stumps: 1.38 ± 0.34/2.35 ± 0.79 mm (P < 0.05); 4) the mean thinnest dimension of the meningeal scar: 0.90 ± 0.43/1.98 ± 0.85 mm (P < 0.05). Conclusion. Vertebral column fixation is a crucial procedure for spinal cord animal experiments.