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Biomedical subjects

S Manek

Publications and source records attributed to S Manek.

At least 55 records · Page 3Linked to original sources
PubMed1996

Effect of mannitol and polyethylene glycol on the action of frusemide during renal storage and transplantation.

Hypoxic injury is a major cause of tubular necrosis in the corticomedullary junction of isolated perfused kidneys, and is ameliorated by inhibitors of active reabsorption, such as frusemide. Our objective was to determine whether frusemide has a similar effect on hypothermically stored transplanted kidneys and whether this effect is modulated by impermeant solutes included in the preservation solution. The effect of frusemide on cytochrome oxidase (cyt aa3) oxidation, renal hemodynamics, and morphology was investigated in the New Zealand White rabbit renal autograft model using near-infrared spectroscopy and light microscopy. A total of 30 kidneys were autografted in six groups. Kidneys were transplanted with or without frusemide either (1) without storage (groups 1 and 2) or after 72 hr of storage in: (2) hypertonic citrate containing mannitol (groups 3 and 4); and (3) hypertonic citrate containing polyethylene glycol (PEG) (groups 5 and 6). In unstored transplanted kidneys, frusemide infusion stimulated a significant (P < 0.05) increase in hemoglobin oxygenation, compared with untreated controls. There was a tendency for cyt aa3 to become reduced, but there were no significant differences between groups 1 and 2. After 72 hr of storage, frusemide infusion stimulated a significant increase in hemoglobin oxygenation in kidneys stored in mannitol (P < 0.005), but there was no significant change in the kidneys stored in PEG. There was a corresponding reduction in cyt aa3 in kidneys stored in mannitol (P < 0.05) but no change in those stored in PEG. These results suggest that frusemide has a significant effect on cortical hemoglobin oxygenation in transplanted kidneys and on active reabsorption in the corticomedullary junction. The selection of impermeant is important and mannitol is significantly superior to PEG in this model.

Animals↗
PubMed1996

Telomerase activity in human breast cancer and benign breast lesions: diagnostic applications in clinical specimens, including fine needle aspirates.

We analysed telomerase activity in normal, benign and malignant breast tissues and in fine needle aspirates by a PCR-based assay. The tissue samples we used in this assay consisted of 20 cryostat sections, 10 microns thick, from each breast biopsy. This method was used to obtain effective extraction from small samples and to confirm the histological identity of the specimen by microscopical examination of serial sections. Fifty-two of 71 breast carcinomas were positive for telomerase activity, and the intensity of this was strong in most cases, whereas all 6 samples of normal breast tissue and 17 of fibrocystic disease were negative and only 1 of 15 fibroadenomas was positive. Invasive ductal carcinomas were more frequently positive than invasive lobular carcinomas. There was no correlation of telomerase activity with tumour size or the occurrence of lymph node metastasis. Evaluation of our assay system showed that a signal of telomerase activity was detectable in extracts from single cryostat sections (< 1 mm2) of a cancer specimen and from as few as 4 cells of a human breast cancer cell line. On the basis of the above data, we applied this assay to fine needle aspirates of breast lesions. Ten of 15 aspirates which had been cytopathologically diagnosed as cancer were strongly positive, while 26 of 29 benign aspirates were totally negative and the remaining 3 showed only borderline activity. In 3 cases, the telomerase result could have helped establish a diagnosis when the cytological observations were inconclusive. Our results indicate that this sensitive assay could become a useful new modality for supplementing microscopic cytopathology in the detection of cancer cells in small tissue biopsies and fine needle aspirates.

Autoradiography↗
PubMed1996

Hemoglobin oxygenation kinetics and secondary ischemia in renal transplantation.

The significance of poor medullary reperfusion in the etiology of acute tubular necrosis during renal transplantation is poorly understood. Our objective was to determine the kinetics of renal hemoglobin oxygenation using near-infrared spectroscopy during renal transplantation, to provide a framework against which the timing of mitochondrial dysfunction could be considered. New Zealand White rabbit kidneys were flushed with hypertonic citrate solution (0-2 degrees C and autografted immediately (group 1) or stored at 0-2 degrees C for 72 hours before autografting (group 2). Changes in oxyhemoglobin (HbO2) and deoxyhemoglobin (Hb) were monitored by near-infrared spectroscopy for 3 hours of reperfusion. Intrarenal perfusion was evaluated separately by barium sulfate angiography. Reperfusion resulted in rapid increases in HbO2 within 1 minute in both groups. Group 1 HbO2 fell sharply to a minimum at 3 minutes but recovered by 20 minutes; group 2 changes were similar, but there was no recovery (P<0.05 by 10 minutes). Hb increased rapidly in both groups upon reperfusion but in group 2 was significantly greater after 10 minutes (P<0.05). Total hemoglobin levels were similar in both groups. Renal hemoglobin saturation was 69% at 1 minute in both groups; there was no significant change in group 1 but a profound desaturation in group 2 to 25% at 10 minute (P<0.005) and no recovery thereafter. Barium sulfate distribution was normal in all group 1 kidneys; cortical distribution was normal in all group 2 kidneys, but medullary perfusion was poor for the first 60 minutes. Renal hemoglobin oxygenation kinetics as determined here do not correlate with the timing of mitochondrial dysfunction previously reported (Thorniley et al., Kidney International, 1994; 45: 1489). We conclude that secondary ischemia during reflow is not the only mechanism leading to acute tubular necrosis.

Animals↗
PubMed1996

The significance of alterations in p53 expression in gynaecological neoplasms.

Current interest in abnormalities of the p53 gene and the abnormal expression of its protein is no less in gynaecological cancer than in the study of neoplasms of other sites. Knowledge of normal p53 gene function (particularly with regard to the regulation of apoptosis) and its derangement through mutation or the influence of viruses is contributing to our understanding of pathogenesis. However, despite the burgeoning amount of new data, critical assessment shows that the required consistency in findings that may have direct clinical application (in terms of prognosis for example) is proving elusive.

Cell Division↗
PubMed1995

Reinnervation and neovascularisation in prefabricated free muscle flaps.

In this study, immediate muscle reinnervation in flap prefabrication was investigated and compared with those flaps which were reinnervated after prefabrication. Using the flow-through wrap-around technique for neovascularisation, denervated external abdominal oblique random muscle flaps in Lewis rats were either immediately reinnervated by implantation of the epigastric nerve or reinnervated late after free transfer, following the prefabrication period of 15 days. Half of the flaps from each group were microsurgically transferred to isogeneic rats. Thirty days later, the flaps were harvested, and neovascularisation and reinnervation were studied with microangiography and immunohistochemistry using antibodies to protein gene peptide (PGP) 9.5, calcitonin gene-related peptide (CGRP), and von Willebrand factor (vWF). Microangiography and quantification of the vWF-stained vessels in the flaps confirmed increased neovascularisation over a longer time course. Reinnervation was found to have no influence over neovascularisation. Nerve sprouting was present 15 days after nerve implantation to the muscle and became widespread by 30 days. CGRP immunoreactivity, which is thought to have some role in trophic and regulatory processes, was observed only after 30 days of reinnervation. Regardless of the timing and extent of reinnervation, a considerable amount of muscle atrophy was observed in the flaps.

Animals↗
PubMed1995

Morphological changes in rat single lung isografts after long-term survival.

A high priority in organ transplantation research is to increase the number of hours that an organ can be successfully preserved. Transplant programmes rely on hypothermia and flush solutions to maintain organ viability during the storage period. We studied long-term morphology in lungs stored for 24 or 48 hours using two modified versions of University of Wisconsin solution, one mimicking the extracellular medium and the other the intracellular medium. Four weeks after transplantation, X-ray and angiograms were used to assess the proportion of ventilating tissue, and light and electron microscopy to analyse morphology. Pulmonary tissue presented near-normal histological appearance in well preserved areas while fibrosis and chronic inflammation were found in scarring processes. Electron microscopy studies revealed some damage-related changes in tissue which appeared histologically normal. Four weeks after transplantation, quality and quantity of recovery were uniform for both solutions tested after 24 hours of storage. However, without reaching significance, after 48 hours the quantity of successfully preserved pulmonary tissue was greater in the group stored in the intracellular solution.

Animals↗
PubMed1994

Inhibition of leukotriene B4 synthesis does not prevent development of acute renal failure following storage and transplantation.

Compound BW B70C, a selective 5-lipoxygenase inhibitor was tested for its ability to reduce inflammatory damage in an in vivo rabbit model of renal storage and transplantation. Kidneys were stored at 0-2 degrees C for 48 hr prior to autografting. In controls, renal vein LTB4 levels rose significantly after 30 min reperfusion but fell after 2 hr to baseline. TxB2 levels remained at baseline for the 6 hr measured. 6-k-PGF1 alpha levels rose significantly after 1 hr of reperfusion and remained elevated thereafter. Histology after 6 hr reperfusion showed moderate-to-severe cortical edema and mild congestion. Infused colloidal carbon was retained in the perivascular area in a narrow band at the corticomedullary junction, indicating a zone of vascular permeability. At 3 days after transplant, kidneys exhibited widespread tubular necrosis and calcification but little inflammation. Serum creatinine and urea peaked between days 3 and 5. 3/6 rabbits showed no symptoms of renal failure after 3 weeks. Pretreatment with BW B70C prevented the increase in LTB4 but had little effect on TxB2 and 6-k-PGF1 alpha levels. Histology showed no amelioration of cortical edema at 6 hr and congestion and hemorrhage were exacerbated. BW B70C had no effect on either colloidal carbon retention or distribution but did significantly reduce tubular necrosis and calcification at day 3. There was very little inflammatory infiltrate. BW B70C treatment did not improve the long-term viability of transplanted kidneys: 2/6 rabbits showed no symptoms of renal failure after 3 weeks. These data indicate that inhibition of LTB4 synthesis by BW B70C does not prevent the development of acute renal failure following 48 hr hypothermic storage and transplantation.

Acute Kidney Injury↗
PubMed1994

Free sensate secondary skin flaps: an experimental study on patterns of reinnervation and neovascularisation.

The main purposes of the present study were: 1) to compare differences, if any, in the patterns of reinnervation between secondary skin flaps, created either from "innervated skin grafts" or from ordinary denervated skin grafts; 2) to examine the influence of time, and the possible roles of granulation tissue and fibrosis in reinnervation following implantation of a nerve trunk into a secondary skin flap at different stages; and 3) to compare neovascularisation before and after free transfer of these flaps. Neurovascular changes were studied in a rat model by microangiography and by immunohistochemical techniques, using antisera to protein gene product 9.5 (panneural marker), to calcitonin gene-related peptide (sensory neurones) and to von Willebrand Factor (endothelial cell marker). The results indicate a potential clinical role for secondary sensate skin flaps where conventional methods of reconstruction including free neurovascular flap transfers are not available.

Animals↗
PubMed1994

An animal model to study the significance of dermis for grafting cultured keratinocytes on full thickness wounds.

Autologous cultured keratinocytes grafted onto full thickness wounds take poorly, and any epidermal cover that is produced is unstable. However, a more stable epidermis has been reported when keratinocytes are grafted onto a dermal surface. We have developed a skin grafting model using a polytetrafluoroethylene (PTFE) skin graft chamber in the domestic pig. The chambers isolate individual wounds and prevent epithelial migration from the wound edge. Dermal grafts were prepared by enzymatic separation of the epidermis from split skin to leave a de-epidermalized dermis (DED). Full thickness wounds were initially grafted with autologous DED and, subsequently, 7 days later with cultured autologous keratinocytes. The wounds were biopsied serially over 6 weeks, and processed for histology, immunocytochemistry and electron microscopy. Clinically, the grafts were seen to mature over the 6-week period. In 14/20 wounds, 40-72 per cent of the wound areas (as assessed by image analysis) had acquired epidermal cover at day 14. In 6/20 wounds, the epidermal cover was 0-27 per cent. The skin surface was stable at day 21. At this time electron microscopy and immunohistochemistry demonstrated a continuous, well formed basement membrane. The epidermis was initially acanthotic, but was histologically mature by day 14. Surprisingly, the dermal grafts were broken down by day 14. However, a neodermis formed beneath all areas with epithelial cover 21 days after grafting the keratinocytes. In this model, we have demonstrated the advantage of providing a dermal bed for cultured keratinocytes.

Animals↗
PubMed1994

Non-invasive measurement of respiratory chain dysfunction following hypothermic renal storage and transplantation.

Ischemia/reperfusion (IR) damage is a major cause of dysfunction in transplanted organs. The objective of the present study was to correlate in vivo measurements of respiratory chain (RC) function with histological and physiological parameters. Non-invasive in situ (surface fluorescence) measurements of mitochondrial NADH and near infrared spectroscopic measurements of cyt aa3 were made in unstored (Group 1) and 72 hour stored (1 to 2 degrees C) (Group 2) autografted rabbit kidneys. The effect of sodium pentobarbitone on NADH levels was investigated. In Group 1, there was a significant change in the redox state of cyt aa3 in all (N = 6) kidneys on reperfusion which correlated with organ viability and increased NADH oxidation and minimal edema on histological examination. In Group 2 there was no significant change in cyt aa3 compared to baseline, and this correlated with poor long term organ viability, slower NADH oxidation, and severe cortical edema. Pentobarbitone inhibition of the RC resulted in rapid and complete reduction of NAD+ in Group 1, but none or only a slight reduction in Group 2. The results demonstrate that it might be possible in future to predict organ viability and histological changes by non-invasive measurements of RC dysfunction in the clinical transplant situation.

Animals↗
PubMed1994

Angiogenesis and reinnervation in skin flaps: the effects of ischaemia examined in an animal model.

In clinical flap transplantation, ischaemia may alter reinnervation patterns either directly or by affecting angiogenesis. This study presents the effects of ischaemia on innervation in totally denervated, transiently (30 minutes) or prolongedly ischaemic skin flaps studied immunohistochemically with antisera to PGP 9.5, CGRP and VWF. Following transient ischaemia, an increase in PGP-immunoreactive (PGP-IR) and CGRP-IR nerve fibres in distant skin by day 12 was followed by increased innervation in immediately adjacent skin. The latter increase was maintained up to 24 days which allowed near normal innervation at the suture margin and in adjacent flap tissue, 0.5 cm from the margin. There was concomitant reinnervation from the pedicle by day 24. In prolongedly ischaemic flaps, an earlier and more prolonged increase in innervation was seen in the entire surrounding skin, with innervation around the suture line at 24 days resembling that in the transiently ischaemic flaps despite initial complete nerve fibre depletion in this area. Hypertrophic nerve fibre clusters were seen in fibrotic areas overlying the pedicle. Vascular changes were similar in both groups with vascularization preceding reinnervation. There were no significant differences in reinnervation between the transiently and prolongedly ischaemic flaps at 24 days, despite considerable initial variations. Ischaemia, CGRP, mediators of chronic inflammation and epidermal factors appeared to stimulate angiogenesis and reinnervation.

Animals↗
PubMed1993

The effect of a synthetic hexadentate iron chelator (CP130) and desferrioxamine on rabbit kidneys exposed to cold and warm ischaemia.

The effect of CP130 (a synthetic hexadentate pyridinone iron chelator) on the formation of two markers of lipid peroxidation (TBA-reactive material and Schiff's bases) in rabbit kidneys following a 72 h period of cold (0-4 degrees C) ischaemia was investigated by either adding CP130 to the flush/storage solution (hypertonic citrate solution) or by administering the agent intravenously 15 min before removal of the organs. In both cases, CP130 blocked the adverse rises in lipid peroxidation caused by ischaemia and subsequent reoxygenation of the homogenates in vitro. Both CP130 and desferrioxamine (DFX) (administered intravenously 15 min before ischaemia and 5 min before reperfusion) were also found to significantly reduce post-ischaemic rates of in vitro lipid peroxidation in kidneys rendered warm ischaemic for 90 min followed by reperfusion for 5 or 60 min in situ. Kidneys exposed to warm ischaemia and reperfusion developed interstitial and intracellular oedema, congestion and haemorrhage. DFX administration had little effect on the histological outcome, whereas CP130 significantly reduced interstitial oedema (at 5 min reperfusion compared to the DFX-treated group), intracellular oedema (at 60 min reperfusion compared to the DFX-treated group) and congestion (at 5 min reperfusion compared with a control group not given any agent). It is concluded that while CP130 and DFX exhibited similar antioxidant properties, CP130 provided better protection from ischaemia/reperfusion injury at the histological level. Synthetic iron chelators may therefore be of benefit in clinical organ transplantation by protecting against tissue damage caused by prolonged ischaemia.

Animals↗
PubMed1993

A porcine model using skin graft chambers for studies on cultured keratinocytes.

In wound healing research, animal models permit an extensive tissue analysis which is not normally possible in clinical studies. A morphological comparison of human and porcine skin was made in order to identify those aspects of the wound healing process where a porcine model may help our understanding of clinical problems. We describe a porcine model for evaluating the growth of cultured keratinocytes on a variety of wound beds. Polytetrafluoroethylene skin graft chambers were used to isolate wounds and prevent epidermal healing from the skin edge. The chambers remained in situ for 5-7 weeks. We detail the surgical technique, the method of porcine keratinocyte culture and highlight some practical measures that were taken to optimise the "take" of the cultured keratinocyte grafts.

Animals↗
PubMed1993

Kerato-dermal grafts: the importance of dermis for the in vivo growth of cultured keratinocytes.

In a porcine model, we studied the benefit of dermis for the growth of cultured autologous keratinocytes (CAK) on full-thickness wounds isolated within skin graft chambers. Kerato-dermal grafts were prepared in a two stage process using autologous de-epidermalised dermis (DED) and CAK (Group 1). Control wounds were prepared by grafting either CAK only (Group 2) or DED only (Group 3). The median epidermal cover of 34 wounds in Group 1 was 47% and was significantly greater (p < 0.001) than the epidermal cover of 12 wounds in Group 2 (4%) and 14 wounds in Group 3 (12%). The epidermis in Group 1 was durable whereas it was fragile in the control wounds. Histologically rête ridges were present at 2 weeks in Group 1, but not in the control wounds. These data indicate that a dermal wound bed significantly improves the in vivo growth of cultured keratinocytes.

Animals↗
PubMed1993

Neovascularisation precedes neural changes in the rat groin skin flap following denervation: an immunohistochemical study.

We have employed immunohistochemical techniques to study neural and vascular changes in rat skin flaps. Following partial or total denervation, flaps were studied at 4, 7 or 12 days using antisera to PGP 9.5 (panneural marker), the neuropeptides CGRP (sensory nerves) and CPON (adrenergic nerves) and an endothelial marker VWF. In partially denervated flaps, moderate increases in PGP-immunoreactive (PGP-IR) and CGRP-IR nerves and a mild increase in CPON-IR nerves in immediate surrounding skin preceded smaller increases in similar nerves around the pedicle. Following total denervation, mild increases in all nerve types at these locations were accompanied by a marked increase of these nerves in distant surrounding skin, 1-2 cm from the suture line. A gradual increase in endothelial cell staining (VWF) of blood vessels was seen in surrounding skin, flap beds and pedicles. Angiogenesis preceded flap reinnervation which occurred initially from surrounding skin, and later from the base, with sensory fibres appearing first.

Animals↗