Regulation of RhoA by STAT3 coordinates glial scar formation.

Understanding how the transcription factor signal transducer and activator of transcription-3 (STAT3) controls glial scar formation may have important clinical implications. We show that astrocytic STAT3 is associated with greater amounts of secreted MMP2, a crucial protease in scar formation. Moreover, we report that STAT3 inhibits the small GTPase RhoA and thereby controls actomyosin tonus, adhesion turnover, and migration of reactive astrocytes, as well as corralling of leukocytes in vitro. The inhibition of RhoA by STAT3 involves ezrin, the phosphorylation of which is reduced in STAT3-CKO astrocytes. Reduction of phosphatase and tensin homologue (PTEN) levels in STAT3-CKO rescues reactive astrocytes dynamics in vitro. By specific targeting of lesion-proximal, reactive astrocytes in Nestin-Cre mice, we show that reduction of PTEN rescues glial scar formation in Nestin-Stat3+/- mice. These findings reveal novel intracellular signaling mechanisms underlying the contribution of reactive astrocyte dynamics to glial scar formation.

Activation and clinical significance of p38 MAPK signaling pathway in patients with severe trauma.

BACKGROUND: Organ dysfunction or multiple organ dysfunction syndrome caused by developing immunological dysfunction and subsequent sepsis or the systemic inflammatory response syndrome after trauma is the leading cause of death in trauma patient. It is believed that mitogen-activated protein kinase) (p38MAPK) is one of the most important kinases in inflammatory signaling. In this study, the change of p38 MAPK signaling pathway in trauma patient with different severity and its clinical significance in trauma inflammation were investigated. METHODS: One hundred fifty major trauma patients were included in the study and divided into three groups according to injury severity score (ISS). All data required to calculate ISS and determine organ function were registered on admission and during the ICU-stay. Peripheral blood samples were collected from trauma patients 6 h, 1 d, 3 d, 5 d, and 7 d after injury. RQ-PCR and Western blot was used to examine the changes in gene expression, protein expression, and activation level of leukocyte p38 MAPK. Plasma IL-6 and TNFalpha were assayed by ELISA. RESULTS: Organ dysfunction in 33 trauma patients developed and eight deaths occurred after 24 h in ICU. The causes of death included severe ARDS, MODS, and irreversible brain injury. Incidence of organ dysfunction was related to the increase of injury severity (P < 0.01). Compared with healthy control, the gene expression of p38 MAPK in trauma patients increased significantly 6 h after injury (P < 0.05), and reached a maximum in 1 d (P < 0.01). The expression maintained a high level for 7 d (P < 0.05). One day after injury, significant elevation was observed in protein expression and activation level of p38 MAPK (P < 0.05), as well as the plasma TNFalpha and IL-6 level (P < 0.01). Further investigation found that the gene expression, protein expression, and activation levels of p38 MAPK increased with higher ISS (P < 0.05), and the elevation of plasma TNFalpha and IL-6 level was associated with the increase of activated p38 MAPK and ISS (P < 0.05). CONCLUSION: p38 MAPK signal pathway was activated in trauma patients. The severity of trauma had highly positive correlation with the expression and activation of p38 MAPK, as well as the elevation of plasma TNFalpha and IL-6 expression. These findings indicate that p38 MAPK signaling pathway plays an important role in the pathological mechanism of trauma.

Regional changes in ectonucleotidase activity after cortical stab injury in rat.

During a variety of insults to the brain adenine nucleotides are released in large quantities from damaged cells, triggering local cellular and biochemical responses to injury. Different models of brain injury reveal that the local increase in adenine nucleotides levels is followed by a compensatory up-regulation of ectonucleotidase enzymes that catalyze sequential hydrolysis of ATP to ADP, AMP and adenosine. However, recent studies imply that changes in adenine nucleotides release may also occur in the areas distant from the site of direct damage. Therefore, in the present study we have used the model of cortical stab injury to analyze extracellular ATP, ADP and AMP hydrolysis in the membrane preparations obtained from the brain regions that were not subjected to direct tissue damage. The brain regions analyzed were contralateral cortex, hippocampus, caudate nucleus, thalamus and hypothalamus. It was evidenced that cortical stab injury induced early widespread decrease in AMP hydrolysis in all brain areas tested, except in the hypothalamus, without changes in ATP hydrolysis. These findings imply that brain injury affects global extracellular adenine nucleotide and nucleoside levels, consequently affecting neuronal function in the regions distant to the primary damage.

Biochemical, functional, and pharmacological characterization of AT-56, an orally active and selective inhibitor of lipocalin-type prostaglandin D synthase.

We report here that 4-dibenzo[a,d]cyclohepten-5-ylidene-1-[4-(2H-tetrazol-5-yl)-butyl]-piperidine (AT-56) is an orally active and selective inhibitor of lipocalin-type prostaglandin (PG) D synthase (L-PGDS). AT-56 inhibited human and mouse L-PGDSs in a concentration (3-250 microm)-dependent manner but did not affect the activities of hematopoietic PGD synthase (H-PGDS), cyclooxygenase-1 and -2, and microsomal PGE synthase-1. AT-56 inhibited the L-PGDS activity in a competitive manner against the substrate PGH(2) (K(m) = 14 microm) with a K(i) value of 75 microm but did not inhibit the binding of 13-cis-retinoic acid, a nonsubstrate lipophilic ligand, to L-PGDS. NMR titration analysis revealed that AT-56 occupied the catalytic pocket, but not the retinoid-binding pocket, of L-PGDS. AT-56 inhibited the production of PGD(2) by L-PGDS-expressing human TE-671 cells after stimulation with Ca(2+) ionophore (5 microm A23187) with an IC(50) value of about 3 microm without affecting their production of PGE(2) and PGF(2alpha) but had no effect on the PGD(2) production by H-PGDS-expressing human megakaryocytes. Orally administered AT-56 (<30 mg/kg body weight) decreased the PGD(2) production to 40% in the brain of H-PGDS-deficient mice after a stab wound injury in a dose-dependent manner without affecting the production of PGE(2) and PGF(2alpha) and also suppressed the accumulation of eosinophils and monocytes in the bronco-alveolar lavage fluid from the antigen-induced lung inflammation model of human L-PGDS-transgenic mice.

Early temporal changes in ecto-nucleotidase activity after cortical stab injury in rat.

During a variety of insults to the brain adenine nucleotides are released in large quantities from damaged cells, triggering multiple cellular responses to injury. Here, we evaluated changes in extracellular ATP, ADP and AMP hydrolysis at different times (0-24 hours) after unilateral cortical stab injury (CSI) in adult rats. Results demonstrated that 24 hours following CSI, ATP and ADP hydrolyzing activities were not significantly altered in injured cortex. Based on calculated V (ATP)/V (ADP) ratio it was concluded that ATP/ADP hydrolysis was primarily catalyzed by NTPDase1 enzyme form. In contrast, AMP hydrolysis, catalyzed by 5'-nucleotidase, was significantly reduced at least 4 hours following CSI. Kinetic analysis and Lineweaver-Burk transformation of the enzyme velocities obtained over the range of AMP concentrations (0.05-1.50 mM) revealed that inhibition of 5'-nucleotidase activity after CSI was of the uncompetitive type. Taken together our data suggest that injured tissue has reduced potential for extracellular metabolism of adenine nucleotides in early stages after CSI.

Lysyl oxidase, the extracellular matrix-forming enzyme, in rat brain injury sites.

Lysyl oxidase is an extracellular enzyme that catalyzes cross-linkages of extracellular matrix proteins. We hypothesized that this enzyme is secreted by cells attracted to central nervous system injury sites and is involved in extracellular matrix modulation and in scar formation. Specific antibodies for immunohistochemistry and enzyme activity measurements were used to detect the presence of lysyl oxidase after longitudinal knife cuts in adult rat forebrain. Immunoreactivity was observed within the core of injury sites from 1 and up to 30 days postoperative, with less staining at 2 and 5 days, and was not associated with glial fibrillary acidic protein-positive astrocytes. Enzyme activity increased transiently in injury site regions with a peak (200% of control) at 10 days postoperative. These results are the first to provide evidence for a time-dependent appearance of active extracellular lysyl oxidase in brain injury sites. They imply that enzyme molecules are synthesized and secreted by cells attracted to brain injury sites and participate in extracellular matrix modulation.

The oligodendroglial reaction to brain stab wounds: an immunohistochemical study.

Myelin/oligodendrocyte specific protein was compared to glial fibrillary acidic protein and 2'3'-cyclic nucleotide 3'-phosphodiesterase expression in normal rat brains and following stab wounds to the cerebral cortex, corpus callosum and hippocampus. Animals with stab wounds were allowed to recover for 5, 15, 28, 45 and 70 days post-operation before fixation by perfusion. Sections were reacted with antibodies against myelin/oligodendrocyte specific protein, glial fibrillary acidic protein and 2'3'-cyclic nucleotide 3'-phosphodiesterase, and observed by light and electron microscopy. Normal cerebral cortex had very few myelin/oligodendrocyte specific protein-positive and 2'3'-cyclic nucleotide 3'-phosphodiesterase-positive cells, but some glial fibrillary acidic protein-positive cells. The myelinated fibres of the corpus callosum were heavily stained for myelin/oligodendrocyte specific protein but unstained by glial fibrillary acidic protein or 2'3'-cyclic nucleotide 3'-phosphodiesterase antibodies. Some immunopositive cells were present in the corpus callosum and hippocampus with all three antibodies. After stab wound myelin/oligodendrocyte specific protein-positive reactive cells had more and longer processes and stained more intensely than equivalent cells in normal brain. These cells were distributed along the wound track, including within the cerebral cortex. The numbers of these cells increased until 28 days post-operation and then decreased so that very few were found at 70 days post-operation except in the corpus callosum. Where demyelination occurred myelin/oligodendrocyte specific protein-staining was lost. Staining for 2'3-cyclic nucleotide 3'-phosphodiesterase revealed a similar pattern. Glial fibrillary acidic protein-positive reactive cells, which were also more robust than the normal cells, were more widely distributed. They increased in number throughout the time periods studied and gliosis was evident on the contralateral side. The glial fibrillary acidic protein-positive astrocytes were also different from the myelin/oligodendrocyte specific protein-positive and 2'3'-cyclic nucleotide 3'-phosphodiesterase-positive oligodendrocytes in terms of cell shape. With electron microscopy myelin/oligodendrocyte specific protein-positive cells showed features typical of immature oligodendrocytes. We conclude that the injury caused a numerical increase in oligodendrocytes and that myelin/oligodendrocyte specific protein is a good marker for the oligodendroglial response and demyelination in pathological conditions.

Localization and quantification of the nonspecific esterase in injured skin for timing of wounds.

Histochemical quantification of the nonspecific esterase (NSE) in injured skin was performed using histochemical demonstration of the enzyme and a microspectrophotometric scanning technique on specimens taken from 32 Hartley guinea-pigs and 8 cases of human skin wounds. In all antemortem incisions and lacerations, including those made at the agonal stage, NSE activity could be observed in the dermal tissue of the wound edge. The enzyme activity increases with the antemortem duration of the injuries. Both total content and mean concentration of NSE in the wound edge between antemortem and postmortem wound groups differ significantly (less than 0.01). Multiple range test shows that significant differences (P less than 0.05 or P less than 0.01) of total content of NSE in the wound edge also exist in 0-5 min, 15-30 min, 1-h, 2-h and 4-h antemortem incised wound groups and in 0-min, 5-min, 15-30 min, 1-h and 4-h antemortem laceration groups. The positive NSE reactions in 8 cases of human skin wounds were similar. The study indicates that histochemical quantification of NSE in injured skin is very useful in timing wounds and is exactly applicable in medicolegal practice. According to the different influences of inhibitors on enzymes, it was inferred that the enzyme activity in wound edges was due to B-esterase.

Fast increase of proteinase inhibitors in necrotic collagenous tissue.

Using the PAP immunohistochemical technique, accumulation of two proteinase inhibitors, alpha-1-antichymotrypsin and alpha-2-macroglobulin, can be detected at the edges of collagenous fibers in the corium after slash wounds of the skin. This accumulation was observed within a survival time of 10-30 min. It, however, is not detectable in postmortally inflicted trauma.

Creatine kinase MB in cases of skeletal muscle trauma.

Fifty-eight patients admitted through our emergency room with severe skeletal muscle injury but no obvious cardiac contusions were evaluated for creatine kinase isoenzyme MB (CK-MB). When such patients show an above-normal value for total CK, it is a question of whether or not myocardial injury has been sustained along with skeletal muscle injury when (a) there are no obvious chest contusions or (b) the patient is unconscious and unable to complain of chest pain. Whenever there is doubt concerning the cardiac status of a patient, lactate dehydrogenase (LD) isoenzymes, serial electrocardiograms, and CK isoenzymes are ordered. Our study revealed that serum of 8.6% of the trauma victims had CK-MB values exceeding 5.0 EU/L (reflecting abnormal CK-MB concentrations) as part of their increased total CK. All patients had normal electrocardiographic patterns along with negative results for LD isoenzymes; none had sustained any demonstrable myocardial injury. The CK-MB value must be interpreted together with the total CK value for appropriate diagnosis in patients with skeletal muscle trauma.

Brain phagocytes: source of high acid phosphatase activity.

A stab wound was produced in the adult rat cerebral cortex, and the progress of enzyme cytochemistry of phagocytosis was studied over 450 days. Light- and electron-microscopic observations were made to establish the origin of high acid phosphatase activity commonly seen at the site of brain lesions. Cells with phagocytic potential became differentiated and activated by the presence of degenerating neurons. The Golgi-lysosomal system of the phagocytes became elaborated, as evidenced by thiamine pyrophosphatase and acid phosphatase activities, the synthesis of acid phosphatase was increased, and the enzyme then secreted into the digestive vacuoles containing dead cells to be digested. Progress of the digestive process resulted in the accumulation of large amounts of acid phosphatase reaction product within the digestive vacuoles. The results showed that the phagocytes were the only detectable source of increased acid phosphatase activity at the site of injury in the cerebral cortex. In contrast to the phagocytes, newly formed multi-nucleated giant cells exhibited weak acid phosphatase, and intense cytochrome oxidase activities, the difference between the two cells reflecting the functional characteristics of each.

[Experimental study of the dynamics of alkaline phsophatase activity after a liver wound]. / Eksperimentalni prouchvaniia vurkhu dinamikata na alkalnofosfataznata aktivnost pri chernodrobna rana.

The investigation of alkaline phosphatase activity on 80 rabbits with an experimental model of liver wound showed the following results: 1. Alkaline phosphatase in blood serum was elevated considerably during the first postoperative days, but after the fifth day was close to the normal ranges. 2. Tissue alkaline phospatase sharply fell up to the third day. Its values began to raise after the 20th day and reached the normal level in the end of the experimental period (30th day). 3. Histochemical studies of liver wound showed a positive reaction for alkaline phosphatase in the cells of the inflammatory infiltrate during the first days. Increased phosphatase activity was demonstrated in fiber structures, epithelium of bile canals and capillary endothelium. The dynamics of alkaline phosphatase activity in blood serum is a reflection of the delicate biochemical and histochemical changes in the liver wound. The follow-up of serum alkaline phosphatase together with the clinical course of healing could be an useful index for evaluation of diet and working capacity of the patient.