Sustainable inflammatory activation following spinal cord injury is driven by thrombin-mediated dynamic expression of astrocytic chemokines.

Acute spinal cord injury (SCI) always results in sustainable recruitment of inflammatory cells driven by sequentially generated chemokines, thereby eliciting excessive neuroinflammation. However, the underlying mechanism of temporally produced chemokines remains elusive. Reactive astrocytes are known to be the main sources of chemokines at the lesion site, which can be immediately activated by thrombin following SCI. In the present study, SCI was shown to induce a sequential production of chemokines CCL2 and CCL5 from astrocytes, which were associated with a persistent infiltration of macrophages/microglia. The rapidly induced CCL2 and later induced CCL5 from astrocytes were regulated by thrombin at the damaged tissues. Investigation of the regulatory mechanism revealed that thrombin facilitated astrocytic CCL2 production through activation of ERK/JNK/NFκB pathway, whereas promoted CCL5 production through PLCß3/NFκB and ERK/JNK/NFκB signal pathway. Inhibition of thrombin activity significantly decreased production of astrocytic CCL2 and CCL5, and reduced the accumulation of macrophages/microglia at the lesion site. Accordingly, the locomotor function of rats was remarkably improved. The present study has provided a new regulatory mechanism on thrombin-mediated sequential production of astrocytic chemokines, which might be beneficial for clinical therapy of CNS neuroinflammation.

HIF-1α promotes astrocytic production of macrophage migration inhibitory factor following spinal cord injury.

BACKGROUND: Macrophage migration inhibitory factor (MIF) is an important mediator of neuropathology in various central nervous system (CNS) diseases. However, little is known about its inducers for production from the nerve cells, as well as the underlying regulatory mechanism. Injury-induced HIF-1α has been shown to exacerbate neuroinflammation by activating multiple downstream target molecules. It is postulated that HIF-1α is involved in the regulation of MIF following spinal cord injury (SCI). METHODS: SCI model of Sprague-Dawley rats was established by cord contusion at T8-T10. The dynamic changes of HIF-1α and MIF protein levels at lesion site of rat spinal cord were determined by Western blot. The specific cell types of HIF-1α and MIF expression were examined by immunostaining. Primary astrocytes were isolated from the spinal cord, cultured and stimulated with various agonist or inhibitor of HIF-1α for analysis of HIF-1α-mediated expression of MIF. Luciferase report assay was used to determine the relationship between HIF-1α and MIF. The Basso, Beattie, and Bresnahan (BBB) locomotor scale was used to assess the locomotor function following SCI. RESULTS: The protein levels of HIF-1α and MIF at lesion site were significantly elevated by SCI. Immunofluorescence demonstrated that both HIF-1α and MIF were abundantly expressed in the astrocytes of the spinal cord. By using various agonists or inhibitors of HIF-1α, it was shown that HIF-1α sufficiently induced astrocytic production of MIF. Mechanistically, HIF-1α promoted MIF expression through interaction with MIF promoter. Inhibition of HIF-1α activity using specific inhibitor markedly reduced the protein levels of MIF at lesion site following SCI, which in turn favored for the functional recovery. CONCLUSION: SCI-induced activation of HIF-1α is able to promote MIF production from astrocytes. Our results have provided new clues for SCI-induced production of DAMPs, which may be helpful for clinical treatment of neuroinflammation.

Heat shock factor 1 promotes neurite outgrowth and suppresses inflammation in the severed spinal cord of geckos.

The low intrinsic growth capacity of neurons and an injury-induced inhibitory milieu are major contributors to the failure of sensory and motor functional recovery following spinal cord injury. Heat shock transcription factor 1 (HSF1), a master regulator of the heat shock response, plays neurogenetic and neuroprotective roles in the damaged or diseased central nervous system. However, the underlying mechanism has not been fully elucidated. In the present study, we used a gecko model of spontaneous nerve regeneration to investigate the potential roles of gecko HSF1 (gHSF1) in the regulation of neurite outgrowth and inflammatory inhibition of macrophages following spinal cord injury. gHSF1 expression in neurons and microglia at the lesion site increased dramatically immediately after tail amputation. gHSF1 overexpression in gecko primary neurons significantly promoted axonal growth by suppressing the expression of suppressor of cytokine signaling-3, and facilitated neuronal survival via activation of the mitogen-activated extracellular signal-regulated kinase/extracellular regulated protein kinases and phosphatidylinositol 3-kinase/protein kinase B pathways. Furthermore, gHSF1 efficiently inhibited the macrophage-mediated inflammatory response by inactivating IkappaB-alpha/NF-kappaB signaling. Our findings show that HSF1 plays dual roles in promoting axonal regrowth and inhibiting leukocyte inflammation, and provide new avenues of investigation for promoting spinal cord injury repair in mammals.

Macrophage Migration Inhibitory Factor Promotes Expression of Matrix Metalloproteinases 1 and 3 in Spinal Cord Astrocytes following Gecko Tail Amputation.

BACKGROUND: The matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases that play a variety of physiological and pathological roles in development, remodeling of tissues and diseases, mainly through degradation of various components of the extracellular matrix (ECM). Particularly, the MMPs have increasingly been found to mediate neuropathology following spinal cord injury (SCI). Proinflammatory mediators are potent activators of the MMPs. However, how the spinal cord regenerative vertebrates circumvent MMPs-mediated neuropathogenesis following SCI remains unclear. METHODS: Following the establishment of gecko tail amputation model, the correlation of MMP-1 (gMMP-1) and MMP-3 (gMMP-3) expression with that of macrophage migration inhibitory factor in gecko (gMIF) was assayed by RT-PCR, Western blot and immunohistochemistry. Transcriptome sequencing of primary astrocytes was performed to analyze the intracellular signal transduction of macrophage migration inhibitory factor (MIF). The effects of MMP-1 and MMP-3 induced by MIF on astrocyte migration were assessed by transwell migration assay. RESULTS: The expression of gMIF significantly increased at lesion site of the injured cord, in parallel with those of gMMP-1 and gMMP-3 in the gecko astrocytes (gAS). Transcriptome sequencing and in vitro cell model revealed that gMIF efficiently promoted the expression of gMMP-1 and gMMP-3 in gAS, which in turn contributed to the migration of gAS. Inhibition of gMIF activity following gecko SCI remarkably attenuated astrocytic expression of the two MMPs, and further influenced gecko tail regeneration. CONCLUSIONS: Gecko SCI following tail amputation promoted production of gMIF, which induced the expression of gMMP-1 and gMMP-3 in gAS. The gMIF-mediated gMMP-1 and gMMP-3 expression was involved in gAS migration and successful tail regeneration.

Thrombin acts as inducer of proinflammatory macrophage migration inhibitory factor in astrocytes following rat spinal cord injury.

BACKGROUND: The danger-associated molecular patterns (DAMPs) are critical contributors to the progressive neuropathology and thereafter affect the functional outcomes following spinal cord injury (SCI). Up to now, the regulatory mechanisms on their inducible production from the living cells remain elusive, aside from their passive release from the necrotic cells. Thrombin is immediately activated by the damaged or stressed central nervous system (CNS), which potently mediates inflammatory astrocytic responses through proteolytic cleavage of protease-activated receptors (PARs). Therefore, SCI-activated thrombin is conceived to induce the production of DAMPs from astrocytes at lesion site. METHODS: Rat SCI model was established by the cord contusion at T8-T10. The expression of thrombin and macrophage migration inhibitory factor (MIF) was determined by ELISA and Western blot. The PAR1, PAR3, and PAR4 receptors of thrombin were examined by PCR and immunohistochemistry. Primary astrocytes were isolated and purified from the spinal cord, followed by stimulation with different concentrations of thrombin either for transcriptome sequencing or for analysis of thrombin-mediated expression of MIF and related signal pathways in the presence or absence of various inhibitors. The post-injury locomotor functions were assessed using the Basso, Beattie, and Bresnahan (BBB) locomotor scale. RESULTS: MIF protein levels were significantly elevated in parallel with those of thrombin induced by SCI. Immunostaining demonstrated that PAR1 receptor, together with MIF, was abundantly expressed in astrocytes. By transcriptome sequencing and bioinformatical analysis of thrombin-stimulated primary astrocytes, MIF was identified to be dynamically regulated by the serine protease. Investigation of the underlying mechanism using various inhibitors revealed that thrombin-activated PAR1 was responsible for the MIF production of astrocytes through modulation of JNK/NFκB pathway. Administration of PAR1 inhibitor at lesion sites following SCI significantly reduced the protein levels of MIF and ameliorated functional deficits of rat locomotion. CONCLUSION: SCI-activated thrombin is a robust inducer of MIF production from astrocytes. Exploring the roles of thrombin in promoting the production of DAMPs from astrocytes at lesion site will provide an alternative strategy for the clinical therapy of CNS inflammation.

[Project to Reduce the Incidence Rate of Nasogastric Tube Slippage in the Rehabilitation Ward].

BACKGROUND: Nasogastric (NG) tubes are used for providing nutrition to patients who have difficulties swallowing. However, the difficult process of repeatedly inserting NG tubes may lead to gastric erosions. PURPOSE: The rehabilitation ward in this study had an NG tube slippage rate of 2.26% in 2012, significantly higher than the same rate in 2011. A proposal was developed to design better techniques for nurses to perform NG tube insertions in order to decrease the rate of NG tube slippage. RESOLUTION: The following were identified as the major causes of NG tube slippage: (1) caretaker is unfamiliar with NG tube care instructions, (2) caretaker disengages NG tube constraints, and (3) improper rotation of patients or carelessness when turning patients. Therefore, the following solutions were proposed: (1) establish an NG tube safety standard operating procedure, (2) publish bilingual (English and Bahasa Indonesia) pamphlets providing instructions on the correct method of securing and placing the NG tubes, (3) make bilingual (English and Bahasa Indonesia) instructional videos showing the correct method for securing and placing the NG tubes, (4) fabricate a novel tube constraint tool (the constraint ball) that is easy to use, (5) install creative bedside NG tube warning signs, and (6) reinforce the importance of NG tube constraint and placement among caretakers. RESULTS: The NG tube slippage rate was reduced from 2.26% to 0.77%, and was maintained at 0.75% during the follow-up period. The caretaker assessment further showed 100% understanding of the implemented NG tube safety procedures. CONCLUSION: This proposal was established using literature research, problem identification, and creative problem solving to achieve an effective reduction in NG tube slippage as well as to enhance nursing quality.

[A project to reduce the incidence of intubation care errors among foreign health aides].

BACKGROUND & PROBLEMS: Foreign health aides are the main providers of care for the elderly and the physically disabled in Taiwan. Correct care skills improve patient safety. In 2010, the incidence of mistakes among foreign health aides in our hospital unit was 58% for nasogastric tube care and 57% for tracheostomy tube care. A survey of foreign health aides and nurses in the unit identified the main causes of these mistakes as: communication difficulties, inaccurate instructions given to patients, and a lack of standard operating procedures given to the foreign health aides. PURPOSE: This project was designed to reduce the rates of improper nasogastric tube care and improper tracheostomy tube care to 20%, respectively. METHODS: This project implemented several appropriate measures. We produced patient instruction hand-outs in Bahasa Indonesia, established a dedicated file holder for Bahasa Indonesian tube care reference information, produced Bahasa Indonesian tube-care-related posters, produced a short film about tube care in Bahasa Indonesian, and established a standardized operating procedure for tube care in our unit. RESULTS: Between December 15th and 31st, 2011, we audited the performance of a total of 32 foreign health aides for proper execution of nasogastric tube care (21 aides) and of proper execution of tracheostomy tube care (11 aides). Patients with concurrent nasogastric and tracheostomy tubes were inspected separately for each care group. The incidence of improper care decreased from 58% to 18% nasogastric intubation and 57% to 18% for tracheostomy intubation. CONCLUSIONS: This project decreased significantly the incidence of improper tube care by the foreign health aides in our unit. Furthermore, the foreign health aides improved their tube nursing care skills. Therefore, this project improved the quality of patient care.