[Effects of transforming growth factor ß1 receptor inhibitor SD-208 on human hypertrophic scar].

OBJECTIVE: To investigate the effects of transforming growth factor ß1 (TGF-ß1) receptor inhibitor SD-208 on human hypertrophic scar and its mechanisms. METHODS: Scar fibroblasts were isolated from deprecated human hypertrophic scar tissue and then sub-cultured. Cells of the fifth passage were used in the following experiments. (1) Cells were divided into blank control group (BC) and 0.5, 1.0, 3.0, and 5.0 µmol/L SD-208 groups according to the random number table (the same grouping method below), with 6 wells in each group. Cells in group BC were added with 1 µL phosphate buffer solution, while cells in the latter four groups were added with 0.5, 1.0, 3.0, and 5.0 µmol/L SD-208, respectively. After being cultured for 12 hours, the proliferation activity of cells was detected by cell counting kit 8 and microplate reader (denoted as absorbance value). Suitable amount of substance concentration of SD-208 according to the results of proliferation activity of cells was chosen for the following experiments. (2) Another batch of cells were divided into group BC and 1, 3 µmol/L SD-208 groups and treated as in (1), with 8 wells in each group. The number of migration cells was detected by transwell method. (3) Another batch of cells were grouped and treated as in (2), and the microfilament morphology of cells was observed by rhodamine-phalloidin staining. (4) Another batch of cells were grouped and treated as in (2), and the protein expression of TGF-ß1 was assessed with Western blotting. (5) Forty-eight BALB/c nude mice were divided into normal saline group (NS) and 1 µmol/L SD-208 group, and one longitudinal incision with length of 1 cm was made on their back. Then human hypertrophic scar tissue was embedded into the incision. On post injury day 7, multipoint injection of NS in a volume of 0.05 mL was performed in wounds of rats in group NS, while rats in 1 µmol/L SD-208 group were given 0.05 mL 1 µmol/L SD-208, once a day. On the day 0 (the same day), 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 post first time of injection, the weight of 8 nude mice was weighed by electronic scale, and scar area was measured by vernier caliper and the ratio of rest scar area was calculated. (6) In week 1, 2, and 3 post first time of injection, the protein expression of TGF-ß1 of human hypertrophic scar tissue was assessed with Western blotting. Data were processed with one-way analysis of variance and two independent-sample t test. RESULTS: (1) The proliferation activity of cells in group BC, 0.5, 1.0, 3.0, and 5.0 µmol/L SD-208 groups was respectively 1.00±0.03, 0.90±0.08, 0.68±0.11, 0.54±0.04, and 0.42±0.09, and the proliferation activity of cells in 0.5, 1.0, 3.0, and 5.0 µmol/L SD-208 groups was significantly lower than that in group BC (with t values from 2.9 to 22.1, P<0.05 or P<0.01). (2) The number of migration cells in 1, 3 µmol/L SD-208 groups was significantly less than that in group BC (with t values respectively 6.5 and 6.4, P values below 0.01). (3) Compared with that in group BC, fluorescence intensity of microfilaments of cells in 1, 3 µmol/L SD-208 groups was attenuated, and the pseudopod extended less. (4) The protein expressions of TGF-ß1 of cells in group BC and 1, 3 µmol/L SD-208 groups were respectively 1.00±0.08, 0.80±0.08, and 0.61±0.05, and the protein expressions of TGF-ß1 of cells in 1, 3 µmol/L SD-208 groups were significantly lower than those in group BC (with t values respectively 4.0 and 9.2, P values below 0.01). (5) The weights of nude mice in group NS and 1 µmol/L SD-208 group were similar on each time day (with t values from 0.2 to 1.1, P values above 0.05). The ratios of rest scar area of nude mice in two groups were decreased along with the injection time, and the ratios of rest scar area of nude mice in 1 µmol/L SD-208 group were significantly less than those in group NS from the day 6 to 20 post first time of injection (with t values from 1.8 to 15.9, P<0.05 or P<0.01). In week 1, 2, and 3 post first time of injection, the protein expressions of TGF-ß1 of human hypertrophic scar tissue in nude mice in two groups showed a tendency of decrease, and the protein expressions of TGF-ß1 of human hypertrophic scar tissue in nude mice in 1 µmol/L SD-208 group were significantly lower than those in group NS (with t values from 6.2 to 19.1, P values below 0.01). CONCLUSIONS: SD-208 has significant inhibition effect on human hypertrophic scars, and the mechanism is correlated to the inhibition of protein expression of endogenous TGF-ß1.

The role of pores in acellular dermal matrix substitute.

To promote the engraftment rate of autologous skin combined with acellular dermal matrix (ADM), ADM was punched to produce regular pores from 500 to 800 µm in diameter, separated by a distance of 3 to 5 mm. The porous ADM was then implanted beneath the flap and transplanted onto an open full-thickness defect wound combined with autografts about 0.2 mm thick in a rat model. The change in diameter of pores in ADM and the neovascularization of ADM matrix were evaluated, and the take rate of porous ADM combined with overlying autologous skin was compared with that of non-porous ADM. The results showed that when porous ADM was grafted onto the full-thickness skin excised wound, plasma penetrated from the wound bed to the surface of ADM through these pores, i.e. the pores punched on ADM were responsible for the imbibition function. Subdermal implantation of ADM indicated that one week post-operation the pores in ADM were still detectable, and some of them contained red blood cells. Two to three weeks after grafting the pores became smaller, partly because of newly synthesized collagen matrix deposition. In Sprague-Dawley rats the engraftment rate of autologous sheet skin graft placed over ADM with pores was 89.5%, which was significantly higher than ADM without pores (63.2%). It is concluded that porous ADM could serve as a good dermal substitute.

FAS deficiency reduces apoptosis, spares axons and improves function after spinal cord injury.

After spinal cord injury (SCI), apoptosis of neurons and oligodendrocytes is associated with axonal degeneration and loss of neurological function. Recent data have suggested a potential role for FAS death receptor-mediated apoptosis in the pathophysiology of SCI. In this study, we examined the effect of FAS deficiency on SCI in vitro and in vivo. FAS(Lpr/lpr) mutant mice and wildtype background-matched mice were subjected to a T5-6 clip compression SCI, and complementary studies were done in an organotypic slice culture model of SCI. Post-traumatic apoptosis in the spinal cord, which was seen in neurons and oligodendrocytes, was decreased in the FAS-deficient mice both in vivo and in vitro particularly in oligodendrocytes. FAS deficiency was also associated with improved locomotor recovery, axonal sparing and preservation of oligodendrocytes and myelin. However, FAS deficiency did not result in a significant increase in surviving neurons in the spinal cord at 6 weeks after injury, likely reflecting the importance of other cell death mechanisms for neurons. We conclude that inhibition of the FAS pathway may be a clinically attractive neuroprotective strategy directed towards oligodendroglial and axonal preservation in the treatment of SCI and neurotrauma.

Transplantation of Composite Skin Containing Keratinocytes Cultured on a Fibroblast-conditioned Acellular Dermal Matrix.

To evaluate the role of fibroblasts in composite skin reconstructed in vitro, four different types of composite skin (A, B, C, and D) were prepared. Human keratinocytes were seeded onto the epidermal side of an acellular dermal matrix (ADM) in type A. Keratinocytes were seeded onto the epidermal side of an ADM and human fibroblasts onto the dermal side in type B. Both keratinocytes and fibroblasts were seeded onto the epidermal side in type C. Type D consisted of fibroblasts on both sides of the ADM and keratinocytes on the epidermal side. The adherence of keratinocytes to the ADM was observed. The composite skin was then transplanted onto full-thickness skin defect wounds in nude mice. Results showed that the adherence of keratinocytes to the ADM was improved when fibroblasts were pre-seeded onto the epidermal side of the ADM. The composite skin was able to close full-thickness skin defect wounds. The take rates were respectively 44.1 ± 7.8%, 47.3 ± 5.4%, 75.2 ± 8.8%, and 81.2 ± 8.1% for types A, B, C, and D. The take rates of types C and D were significantly higher than those of types A and D. There was no significant difference in take rate between types C and D. In conclusion, composite skin consisting of keratinocytes cultured on a fibroblast-conditioned ADM was a good skin substitute.

Oligodendroglial apoptosis occurs along degenerating axons and is associated with FAS and p75 expression following spinal cord injury in the rat.

Apoptosis or programmed cell death has been reported after CNS trauma. However, the significance of this mechanism in the pathophysiology of spinal cord injury, in particular at the cervical level, requires further investigation. In the present study, we used the extradural clip compression model in the rat to examine the cellular distribution of apoptosis following cervical spinal cord injury, the relationship between glial apoptosis and post-traumatic axonal degeneration and the possible role of apo[apoptosis]-1, CD95 (FAS) and p75 in initiating post-traumatic glial apoptosis. In situ terminal-deoxy-transferase mediated dUTP nick end labeling revealed apoptotic cells, largely oligodendrocytes as identified by cell specific markers, in grey and white matter following spinal cord injury. Apoptotic cell death was confirmed using electron microscopy and by the demonstration of DNA laddering on agarose gel electrophoresis. Beta-amyloid precursor protein was used as a molecular marker of axonal degeneration on western blots and immunohistochemistry. Degeneration of axons was temporally and spatially co-localized with glial apoptosis. FAS and p75 protein expression was seen in astrocytes, oligodendrocytes and microglia, and was also seen in some apoptotic glia after cord injury. Both FAS and p75 increased in expression in a temporal course, which mirrored the development of cellular apoptosis. The downstream caspases 3 and 8, which are linked to FAS and p75, demonstrated activation at times of maximal apoptosis, while FLIP-L an inhibitor of caspase 8, decreased at times of maximal apoptosis. We conclude that axonal degeneration after traumatic spinal cord injury is associated with glial, in particular oligodendroglial, apoptosis. Activation of the FAS and p75 death receptor pathways may be involved in initiating this process.

Systemic hypothermia following spinal cord compression injury in the rat: an immunohistochemical study on MAP 2 with special reference to dendrite changes.

Systemic hypothermia has been shown to exert neuroprotective effects in experimental ischemic CNS models caused by vascular occlusions. The present study addresses the question as to whether systemic hypothermia has similar neuroprotective qualities following severe spinal cord compression trauma using microtubule-associated protein 2 (MAP2) immunohistochemistry combined with the avidin-biotin-peroxidase complex method as marker to identify neuronal and dendritic lesions. Fifteen rats were randomized into three equally sized groups. One group sustained thoracic laminectomy, the others severe spinal cord compression trauma of the T8-9 segment. The control group contained laminectomized animals submitted to a hypothermic procedure in which the esophageal temperature was reduced from 38 degrees C to 30 degrees C. The two trauma groups were either submitted to the same hypothermic procedure or kept normothermic during the corresponding time. All animals were sacrificed 24 h following the surgical procedure. The MAP2 immunostaining in the normothermic trauma group indicated marked reductions in MAP2 antigen in the cranial and caudal peri-injury zones (T7 and T10, respectively). This reduction was much less pronounced in the hypothermic trauma group. In fact, the MAP2 antigen was present in almost equally sized areas in both the hypothermic groups independent of previous laminectomy alone or the addition of trauma. Our study thus indicates that hypothermia has a neuroprotective effect on dendrites of rat spinal cords subjected to compression trauma.

A metabolic threshold of irreversible ischemia demonstrated by PET in a middle cerebral artery occlusion-reperfusion primate model.

OBJECTIVE: to evaluate the predictive value of measurements of regional cerebral blood flow (CBF), oxygen metabolism (CMRO2) and oxygen extraction ratio (OER) for assessment of the fate of ischemic brain tissue. MATERIALS AND METHODS: Sequential PET measurements were performed during middle cerebral artery occlusion (MCAO; 2 h) and 12-24 h (mean 18 h) of reperfusion in a primate model (Macaca mulatta, n = 8). A penumbra region was delineated on the MCAO PET image (OER > 125% and CMRO2> or = 45% of the values observed in the contralateral hemisphere, respectively) and an infarction region was delineated on the last PET image (CMRO2 <45% of the values observed in the contralateral hemisphere). The penumbra regions delineated during MCAO and the infarction regions delineated at the final PET, were copied on to the images from all other PET sessions for measurements of CBF, CMRO2 and OER. Ratios were calculated by dividing the mean values obtained by the values of the corresponding contralateral region. RESULTS: Histopathology verified the adequacy of the criteria applied in the last PET for delineation of the infarction region. The penumbra region and infarction region were separated in all cases, except in two cases where a minimal overlap was seen. CBF and OER showed considerable variation over time and there was no consistent difference between the penumbra and infarction regions. CMRO2 showed a more stable pattern and the difference between penumbra and infarction regions was maintained from the time of MCAO throughout the entire reperfusion phase. With CMRO2 as predictor, all 50 observations could be correctly predicted as penumbra or infarction when using an optimal threshold ratio value estimated to be in the interval of 61% to 69% of the corresponding contralateral region. CBF and OER proved to have low power as predictors. CONCLUSIONS: The results indicate that CMRO2 is the best predictor of reversible or irreversible brain damage and the critical metabolic threshold level appears to be a reduction of oxygen metabolism to between 61% and 69% of the corresponding contralateral region.

Systemic hypothermia following spinal cord compression injury in the rat: axonal changes studied by beta-APP, ubiquitin, and PGP 9.5 immunohistochemistry.

STUDY DESIGN: Systemic hypothermia exerts neuroprotective effects in experimental ischemic CNS models caused by vascular occlusions. Recent experimental and clinical studies have also demonstrated beneficial effects of hypothermic treatment following brain trauma. OBJECTIVES: The present study addresses the question as to whether systemic hypothermia has similar protective qualities following severe spinal cord compression trauma using beta-APP-, ubiquitin-, and PGP-9.5-immunohistochemistry combined with the ABC complex method as markers to identify axonal changes. METHODS: Fifteen rats were randomized into three equally large groups and sustained to either thoracic laminectomy or to severe spinal cord compression trauma of the Th 8 - 9 segments. The non-trauma group contained laminectomized animals submitted to a hypothermic procedure in which the core temperature was reduced from 38 to 30 degrees C. The two trauma groups were either submitted to the same hypothermic procedure or kept normothermic during the corresponding time. All animals were sacrificed 24 h following the surgical procedure. RESULTS: In the hypothermic non-trauma group no axonal changes were seen. The number of abnormal axons, as indicated by accumulation of immunoreactive material in enlarged axons, was lower in the peri-injury zones of the hypothermic trauma group than in the normothermic trauma group. This difference was most obvious in the cranial peri-injury zones. No differences were seen between the groups in the trauma zones. CONCLUSIONS: This study demonstrates reduced axonal swelling in the peri-injury zones of spinal cord injured rats treated with systemic hypothermia. These changes could either indicate neuroprotective effects of the hypothermic treatment, or be results of reduced axonal transport or protein synthesis. To evaluate the clinical importance of our findings, further studies including reliable outcome measures of the animals must be performed.

Systemic hypothermia following compression injury of rat spinal cord: reduction of plasma protein extravasation demonstrated by immunohistochemistry.

Systemic hypothermia has neuroprotective effects in experimental models of central nervous system ischemia caused by vascular occlusions. The present study addresses the question as to whether systemic hypothermia can influence the extravasation of plasma proteins following severe spinal cord compression trauma using immunohistochemistry to identify the plasma proteins albumin, fibrinogen and fibronectin. Fifteen rats were assigned to one of three groups and received either thoracic (T) laminectomy or severe spinal cord compression trauma of the T8-9 segment. One group comprised laminectomized animals without compression trauma submitted to a hypothermic procedure in which the core temperature was reduced from 38 degrees to 30 degrees C. The two trauma groups were either submitted to the same hypothermic procedure or kept normothermic during the corresponding time. All animals were killed 24 h following the surgical procedure. The normothermic and hypothermic trauma groups had indications of marked extravasation of albumin, fibrinogen and fibronectin at the site of the injury (T8-9). There was also pronounced extravasation in the cranial and caudal peri-injury zones (T7 and T10) of normothermic injured rats but, with few exceptions, not in the hypothermic ones with the same degree of compression. By measuring the cross-sectional area of the peri-injury zones we found in the hypothermic trauma group a significant reduction of the expansion compared with that present in normothermic injured rats. Our study thus indicates that hypothermia reduces the extravasation of the plasma proteins albumin, fibrinogen and fibronectin following spinal cord compression in the rat. Such a reduction may contribute to neuroprotective effects exerted by hypothermia.

Systemic hypothermia after spinal cord compression injury in the rat: does recorded temperature in accessible organs reflect the intramedullary temperature in the spinal cord?

This article addresses one basic issue regarding the use of systemic hypothermia in the acute management of spinal cord injury, namely, how to interpret temperature recordings in accessible organs such as the rectum or esophagus with reference to the spinal cord temperature. Thirty-six rats, divided into six groups, were randomized to laminectomy or to severe spinal cord compression trauma, and were further randomized to either a cooling/rewarming procedure or continuous normothermia (esophageal temperature 38 degrees C) for 90 min. The first procedure comprised normothermia during the surgical procedure, followed by lowering of the esophageal temperature from 38 degrees C to 30 degrees C (the hypothermic level), a 20-min steady-state period at 30 degrees C, rewarming to 38 degrees C, and finally a 20-min steady-state period at 38 degrees C. The esophageal, rectal, and epidural temperatures were recorded in all animals. The intramedullary temperature was also recorded invasively in four of the six groups. We conclude that the esophageal temperature is safe and easy to record and, in our setting, reflects the epidural temperature. The differences registrated may reflect a true deviation of the intramedullary temperature due to initial environmental exposure and secondary injury processes. Our results indicate that the esophageal temperature exceeds the intramedullary temperature during the initial recording and final steady state following rewarming, but not during the most crucial part of the experiment, the hypothermic period. The core temperature measured in the esophagus can therefore be used to evaluate the intramedullary temperature during alterations of the systemic temperature and during hypothermic periods.

Regulation of gp330/megalin expression by vitamins A and D.

BACKGROUND: A membrane-bound 550-kD Ca2+-binding glycoprotein belonging to the low-density lipoprotein (LDL) receptor superfamily has recently been identified as a putative calcium-sensing molecule. This molecule, known as gp330/megalin, is among several tissues present in the proximal tubule, parathyroid and placental cytotrophoblasts, in which a Ca2+-sensing function has been demonstrated. METHODS: Regulation of mRNA and protein expression of gp330/megalin were studied in a recently established cell line derived from rat kidney proximal tubule cells (IRPTCs), in human JEG-3 cells and in the mouse embryonal carcinoma cell line F9. RESULTS: In IRPTCs, quantification of mRNA and protein expression demonstrated two- to five-fold increases after addition of 10(-6) mol L(-1) all-trans-retinoic acid, 9-cis-retinoic acid or 1,25-dihydroxyvitamin D3, alone or in combination. Similarly, an increase in gp330/megalin mRNA expression was seen in JEG-3 cells cultured with vitamin D and retinoids, as well as when F9 cells were differentiated by incubation with retinoic acid and cAMP. The IRPTCs were immortalized by viral infection with the SV40 genome preceded by a temperature-sensitive promoter. Thus, by culture of the cells at 41 degrees C, SV40 genome transcription is inhibited and the IRPTC phenotype is reversed towards non-infected proximal tubule cells. At 41 degrees C, gp330/megalin mRNA expression was significantly increased compared with cells incubated at 34 degrees C. CONCLUSION: The results indicate a correlation between exposure to retinoic acid or vitamin D or induction of cell differentiation (by retinoic acid/cAMP in F9 cells or inhibition of SV40 transcription in IRPTCs) and an increase in gp330/megalin protein and mRNA expression.

Accumulation of immunoreactivity to ubiquitin carboxyl-terminal hydrolase PGP 9.5 in axons of human cases with spinal cord lesions.

The protein gene product PGP 9.5 is one of the major polypeptides in neurons. It can act as a ubiquitin carboxyl-terminal hydrolase in ubiquitin-mediated degradation of proteins. The present study was performed to find out if human cases with spinal cord trauma present immunohistochemical signs of PGP 9.5 accumulation in injured axons known to accumulate ubiquitin. For comparison, we used six autopsy cases without spinal cord pathology, one case with syringomyelia, one case with ischaemic injury of the cord, and six ALS cases. Controls presented PGP 9.5-immunostained axons of weak to moderate intensity in the longitudinal tracts. Immunoreactivity was not detected in nerve cell bodies, glial cells or axons of the grey matter. All nine trauma cases showed axonal swellings, but their numbers varied. Intensely immunostained axonal swellings were particularly abundant in cases with a survival period up to 1 month after trauma. Strongly immunoreactive axons were present also in the cases with infarct and syringomyelia. In conclusion, human cases with spinal cord trauma and other focal injuries present signs of PGP 9.5 accumulation in severed axons possibly resulting from disturbed axonal transport. PGP 9.5 thus seems to be present and may take part in ubiquitin-mediated degradation of proteins in injured axons of the spinal cord.

Biosynthesis and function of all-trans- and 9-cis-retinoic acid in parathyroid cells.

We demonstrate that cultured human and bovine parathyroid cells incubated with all-trans-[11,12-3H]-retinol convert this tracer into all-trans- and 9-cis-retinoic acid. By using RT-PCR, cellular retinol-binding protein type I (CRBP I), cellular retinoic acid binding protein I and II (CRABP I and II), retinoic acid receptors (RARs) alpha, beta and gamma, and 9-cis-retinoic acid receptor (RXR) alpha transcripts were detected in human parathyroid cDNA. CRBP I and CRABP I expression was confirmed by immunohistochemistry. Both 9-cis- and all-trans-RA were found to suppress parathyroid hormone (PTH) secretion from dispersed human adenomatous parathyroid cells, which was augmented by combined treatment with 1mM RA and 100 nM 1,25 (OH)2D3. The present data establish parathyroid gland as a target for retinoids and as a site of synthesis of the hormonal forms of vitamin A (retinol), all-trans- and 9-cis-retinoic acid.

XY-quadrivalent association and sterility in a man carrier of a reciprocal autosomal translocation involving the whole arm of an acrocentric chromosome t(2;15)(q21.3;cen).

Meiotic and synaptonemal complex studies using electron microscopy were carried out on an infertile man with a 46,XY t(2q;15p). Synaptonemal complex analysis showed terminal asynapsis in the totality of quadrivalents and a high and significant frequency of association with the XY vesicle (80%), possibly related to the high amount of satellite DNA of the acrocentric chromosome 15. In this translocation carrier, the XY quadrivalent association at pachytene stage is positively correlated with the degree of spermatogenic breakdown after pachytene stage. Whether association with the non-paired segment represents the causative factor or only a secondary effect has still to be clarified.