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PubMed1988

Kinetics of development of inflammatory lesions in myocardial and skeletal muscle in experimental Trypanosoma cruzi infection.

We studied the kinetics of development of inflammation in the myocardium and skeletal muscles of mice infected with Trypanosoma cruzi by determining the numbers of mononuclear cells (MNC), neutrophils, and eosinophils at tissue sites with varying degrees of damage. In the myocardium, areas with incipient inflammation and preserved tissue had the smallest numbers of inflammatory cells, 96-100% of which were MNC. In lesions where inflammatory cells accumulated in interstitial spaces displacing myofibers, MNC were also predominant (greater than 98%) but were present in larger numbers than in areas with preserved tissue. The number of MNC was even larger in necrotic areas where there was also marked neutrophil infiltration at the time when amastigote nests were frequently present. In skeletal muscle, MNC were also the first cells to infiltrate lesion sites; their numbers increased with the degree of severity of the lesion. Neutrophil accumulation also accompanied skeletal muscle necrosis. A salient difference was eosinophil accumulation in the necrotic lesions of skeletal muscle but not in the myocardium. The results identify MNC as the cell that initiates the inflammatory process in the heart and skeletal muscles of T. cruzi-infected mice. In these tissues the number of MNC appeared to be a good correlate of lesion severity.

Animals↗
PubMed1994

Protective effect of preservation of canine pancreas by the two-layer (University of Wisconsin solution/perfluorochemical) method against rewarming ischemic injury during implantation.

Rewarming ischemia during implantation severely compromises posttransplant pancreas graft survival because the graft has already been subjected to warm and cold ischemia before implantation. The purpose of this study was to examine whether preservation of the pancreas graft by the two-layer method ameliorates rewarming ischemic injury of the graft during implantation using a canine model. After flushing with cold University of Wisconsin solution (UW), the pancreas grafts were preserved by the two-layer (UW/perfluorochemical [PFC]) method (group 1) or simple cold storage in UW (group 2) for 24 hr and then autotransplanted. In control, the pancreas grafts were flushed out with cold UW and immediately autotransplanted without preservation (group 3). After completion of vascular anastomosis, vascular clamp was not released until 90, 120, or 150 min of rewarming ischemia, including anastomosis time, had elapsed. After 90 min of rewarming ischemia, graft survival rates were 5/5, 100%, 5/5, 100%, and 5/5, 100%, in groups 1, 2, and 3, respectively. After 120 min, all the grafts in groups 2 and 3 failed (0/5, 0%, and 0/5, 0%, respectively); however, all the grafts in group 1 survived (5/5, 100%). Even after 150 min, 1 of 3 grafts in group 1 survived (1/3, 33%). After 24 hr preservation, tissue ATP levels of the grafts in group 1 were about 2-fold the reference values before harvesting (8.23 +/- 0.72 vs. 4.44 +/- 0.49 mumol/g dry weight, P < 0.05) and significantly higher compared with group 2 (8.23 +/- 0.72 vs. 1.76 +/- 0.52 mumol/g dry weight, P < 0.01). After 120 min of rewarming ischemia, tissue ATP levels in group 1 were 84% of the reference values and significantly higher compared with group 2 (3.75 +/- 0.25 vs. 1.57 +/- 0.48 mumol/g dry weight, P < 0.05). Two hours after reperfusion, ATP levels in group 1 were 42% of reference values but significantly higher compared with group 2 (1.86 +/- 0.36 vs. 1.03 +/- 0.18 mumol/g dry weight, P < 0.05). We conclude that the two-layer (UW/PFC) method ameliorates rewarming ischemic injury of the pancreas graft during implantation by increasing tissue ATP contents during preservation and consequently maintaining tissue ATP levels during implantation.

Adenosine↗
PubMed2001

Electron microscopic assessment of smeared or imprinted specimens of solid tumours.

The aim of this study was to develop a reliable method for preparation of smeared or imprinted cytology specimens for electron microscopic examination. Ten different solid tumours were studied. In each case one air-dried (Diff-Quik stained) and one alcohol-fixed (Pap stained) smear was prepared for diagnostic purposes. Simultaneously, a third smear or imprint was prepared for electron microscopy on a coverslip that was coated with poly-L-lysine and attached to a glass slide using double-sided adhesive tape. The smear or imprint was primary fixed in 2% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, for 2 hours. The coverslip was removed from the slide, placed in a glass petri dish and processed for electron microscopy. The smear/imprint on the coverslip was embedded in resin on a silicon embedding mould and allowed to polymerise for 8-12 hours. The coverslip was removed using liquid nitrogen and the block sectioned using standard techniques as for a monolayer. The specimens collected for electron microscopy using this technique yielded sufficient material for assessment with excellent tissue preservation producing good ultrastructural detail. Focal mechanical damage was seen in some specimens but diagnostic areas were always found within the block. As the smear/imprint can be regarded as a monolayer, the processing time can be reduced compared with solid tissue specimens. This technique ensures that well-preserved tissue is available for electron microscopy even when the sample size is very limited.

Biopsy, Needle↗
PubMed2004

Metabolic mechanism by which mild regional hypothermia preserves ischemic tissue.

BACKGROUND: Our laboratory demonstrated that mild regional hypothermia reduced myocardial infarct size by an average of 65% in the rabbit model of regional ischemia. The exact mechanism for this benefit has not been explored. We hypothesized that a moderate reduction in regional myocardial temperature could preserve cardiac energy metabolism and thus protect the myocardium from sustained ischemic insult. METHODS AND RESULTS: Anesthetized open-chest rabbits were randomized to normothermic sham-operated (NS, n = 6), hypothermic sham-operated (HS, n = 6), normothermic ischemic (NI, n = 10), and hypothermic ischemic (HI, n = 10) groups. Both sham-operated groups received no occlusions, and both ischemic groups were subjected to 20 minutes of coronary occlusion. To achieve regional cooling of the hearts in the hypothermic groups, a bag of ice water was placed directly on the risk area 15 minutes prior to coronary artery occlusion/no intervention and maintained for the duration of the subsequent 20 minutes of ischemia/no intervention (in the HI and HS groups respectively). Hypothermia preserved adenosine triphosphate (ATP) and glycogen stores in the ischemic area by 42.9% and 84.2%, respectively (1.20 +/- 0.11 micromoles ATP/g wet tissue vs 0.84 +/- 0.06 micromoles ATP/g wet tissue and 8.16 +/- 0.95 micromoles of glucosyl unit/g wet tissue vs 4.43 +/- 0.44 micromoles of glucosyl unit/g wet tissue in the HI and the NI groups, respectively). In addition, hypothermia resulted in a trend toward creatine phosphate preservation in the nonischemic area. CONCLUSIONS: This is the first demonstration that local therapy with mild reductions in myocardial temperature preserves energy metabolism both in the ischemic and the nonischemic areas as well. The preservation in ATP is the likely mechanism by which regional hypothermia is preserving ischemic myocardium.

Adenosine Triphosphate↗
PubMed2003

Preservation of vascular tissue under hypothermic conditions.

BACKGROUND: Preservation of vascular tissue plays a crucial role in the success of organ transplantation. We investigated and compared the performance of 4 preservation solutions at preserving vascular tissue over 24, 48, and 72 h under hypothermic conditions. MATERIALS AND METHODS: Rat aortic segments were stored in University of Wisconsin (UW), Modified University of Wisconsin (mUW), EuroCollins (EC), and Bretschneider Histidine-tryptophan-ketoglutarate (HTK) solutions at 4 degrees C for 24, 48, and 72 h. Functional assessment was performed by measuring smooth muscle cell contraction and endothelium-dependent relaxation after stimulation with phenylephrine and acetylcholine, respectively, in an organ bath. RESULTS: UW and EC solutions were superior to the other solutions for endothelial cell preservation and were not statistically different from each other at any time (P < 0.05). Smooth muscle cell (i.e., contractile) preservation was best achieved with UW solution for up to 72 h. HTK appeared to function well at 24 h, but at 48 and 72 h, there was a major decline in contractile response and relaxation. CONCLUSION: UW solution appears to be superior for the preservation of the functional integrity of vascular tissue stored under hypothermic conditions up to 72 h.

Animals↗
PubMed2001

The role of frozen storage in preserving adipose tissue obtained by suction-assisted lipectomy for repeated fat injection procedures.

BACKGROUND: The injection of autologous free fat obtained by suction-assisted lipectomy for the correction of soft tissue defects is a common procedure in plastic surgery. However, unpredictable partial absorption of the injected fat often necessitates repeated procedures. OBJECTIVE: To examine the role of frozen storage as a means of preserving the fat obtained by suction-assisted lipectomy for repeated procedures. METHODS: Human adipose tissue obtained by suction-assisted lipectomy was stored in a domestic refrigerator at -18 degrees C for 2 weeks. After thawing, the fat was injected into nude mice. In the control group, the fat was injected immediately after the harvesting procedure. Grafts were dissected out and compared 15 weeks postinjection. RESULTS: Injected fat survived in both study and control groups. No significant differences were found between fat graft weight and volume, or in any of the histologic parameters examined. CONCLUSION: Fat obtained by suction-assisted lipectomy may be preserved for future use by freezing.

Adipose Tissue↗
PubMed1997

A fixation procedure for ultrastructural investigation of synaptic connections in resected human cortex.

Electron microscopic investigations of the fine circuitry of human central nervous system require a well-preserved tissue ultrastructure. Because the deterioration of subcellular structures occurs rapidly in postmortem human brain, the use of a fixation by immersion of surgically resected human nervous tissue would be advantageous to investigate directly its synaptic circuitry. To obtain an optimal preservation of subcellular elements in immersion-fixed brain tissue, different conditions of fixation were first tested on 400 microns-thick sections of rat neocortex. Parameters tested were temperature of the fixative solution, concentrations of glutaraldehyde and of cacodylate buffer with or without microwave irradiation, and finally, the presence of dimethyl sulfoxide. The best ultrastructural preservation was obtained by immersing the tissue in 0.1 M cacodylate buffer, 3.0 mM CaCl2, 2% paraformaldehyde, 2.5% glutaraldehyde, and 2.5% dimethyl sulfoxide at 37 degrees C for 5 min and then at 4 degrees C for 4 h. This procedure of fixation was then applied to human neocortical tissue resected to alleviate temporal lobe epilepsy. This method led to good tissue preservation in addition to retaining the antigenicity to the inhibitory amino acid neurotransmitter, gamma-aminobutyric acid (GABA). Therefore, the tissue preservation obtained would permit these chemically defined connections to be investigated quantitatively at the electron microscopic level in resected human cortex.

Animals↗
PubMed2003

Vortex disaggregation for flow cytometry allows direct histologic correlation: a novel approach for small biopsies and inaspirable bone marrows.

BACKGROUND: Many approaches to obtaining single cells from tissue for flow cytometric immunophenotyping are used; however, these methods result in tissue that is too disrupted for subsequent histologic examination. We introduce a new technique for cell dissociation of hematopoietic malignancies that preserves tissue for histology. This is especially important with small specimens for which this type of correlation is critical. METHODS: Fresh tissue from lymph node, gastrointestinal (GI) tract, skin, and other soft tissue biopsies, in addition to cores of inaspirable bone marrows, were briefly vortexed until the RPMI cell culture medium became cloudy. Larger specimens such as lymph nodes were sectioned before disaggregating, whereas smaller ones were vortexed in toto. Resultant flow cytometric analyses were compared with the histology and, in some cases, the immunohistochemistry (IHC) to determine whether the data were concordant. Cell suspensions of 104 specimens-composed of 48 lymph nodes, 19 bone marrow cores (BMCs), 11 GI biopsies, 11 skin/soft tissue biopsies, and 15 miscellaneous specimens-were prepared via vortex disaggregation. RESULTS: Flow cytometric analysis of 96 specimens (92.3%) showed adequacy of material and diagnostic correlation with the histology and IHC. Of the eight cases (7.7%) that were discordant, seven were attributable to significant specimen fibrosis or necrosis. With respect to tissue type, this method produced diagnostic cell suspensions for most lymph nodes (95.8%), GI biopsies (90.9%), and BMCs (89.5%); however, it was less useful for skin/soft tissue samples (81.8%). CONCLUSIONS: Disaggregation of tissue for flow cytometric analysis by vortexing appears to provide adequate and representative cellular material. This technique is ideal for inaspirable bone marrows and small biopsies where tissue preservation for histology is paramount.

Biopsy↗
PubMed1998

Detection of the sex-determining region of the Y chromosome in 46,XX true hermaphroditism.

OBJECTIVES: Two cases of 46,XX true hermaphroditism were analyzed for two Y-DNA sequences, including the recently cloned gene for male testis determination, the sex-determining region of the Y chromosome (SRY). METHODS: Polymerase chain reaction was performed to amplify the SRY. DNA was prepared from peripheral blood lymphocytes as well as from gonadal tissue preserved in a paraffin block. RESULTS: One hermaphrodite contained the SRY sequences in peripheral blood lymphocytes and the testicular part of ovotestis tissue preserved in a paraffin block, while in the second patient these sequences were not detected. CONCLUSIONS: The SRY positive subject resulted from occult Y mosaicism rather than from X-Y translocation. Testis differentiation in the SRY negative subject may have been caused by mutation of a gene on the X chromosome or, alternatively, on an autosome.

Disorders of Sex Development↗
PubMed2003

[Overwhelming postsplenectomy infection].

BACKGROUND: Splenectomy performed at any age and for any reason increases the risk for death due to overwhelming infection. AIM: To evaluate definition, etiology, incidence, risk factors and prophylaxis of overwhelming postsplenectomy infection, as well as the methods related to splenic tissue preservation when total splenectomy is necessary. METHOD: Bibliographic review. RESULTS: The etiological agents more frequently found are Streptococcus pneumoniae, Haemophilus influenza and type B, and Neisseria meningitidis. Other bacteria like Escherichia coli, Streptococcus b-hemolytic, Staphylococcus aureus and Pseudomonas sp represent a significant risk as well. In addition, a great variety of agents including other enteric Gram-negative microorganisms and non-bacteria pathogens can also be sporadically identified. The prophylaxis is based on three main aspects: patient's education, immune prophylaxis and chemical prophylaxis. However these are not enough to prevent the higher risk of developing sepsis. When total splenectomy is unavoidable, heterotopic splenic autotransplantation seems to be the only alternative for splenic tissue preservation. According to clinical and experimental studies, the splenic autotransplanted tissue present a similar structure to a normal spleen and preserve the splenic immune function. CONCLUSIONS: The high risk of overwhelming postsplenectomy infection, reduced the indication for total splenectomy, in trauma and several diseases. Prophylactic methods have been developed to minimize the effects of the sepsis. Several researches have been done to determine the immunocompetence of autogenous splenic grafts in response to bacteria invasion.

Bacterial Infections↗
PubMed2002

Noncryogenic preservation of mammalian tissues for DNA extraction: an assessment of storage methods.

Reliable field methods for the storage of tissues to be used for DNA extraction and amplification are critical to many studies employing molecular techniques. Protection from DNA degradation was compared among three commonly used methods of noncryogenic storage of tissues over a time scale of 2 years. All three methods prevented DNA degradation during storage for at least 6 months. DMSO (dimethyl sulfoxide)-salt solution provided the best protection from DNA degradation of tissues stored for up to 2 years. High molecular weight DNA was recovered from lysis buffer in which tissue was stored for 2 years, however, moderate amounts of degraded DNA was also present. High molecular weight DNA was recovered from tissues stored in ethanol for 2 years, however the yield was relatively small compared to the other two noncryogenic storage techniques. Much of the DNA degradation in ethanol preserved tissues appeared to occur during the extraction procedure and can be reduced by soaking the tissue in lysis buffer for a few hours prior to beginning the extraction. The yield of PCR products was greatest from DNA extracted from DMSO-salt solution preserved tissues, whereas DNA from tissues stored in either lysis buffer or ethanol produced lower yields.

Animals↗
PubMed2002

Postmortem RNA and protein stability in perinatal human lungs.

The availability of fetal and neonatal lung tissue is an invaluable resource to elucidate the molecular regulation of human lung development. In this study, we have investigated the mRNA and protein stability of perinatal lung tissues treated with RNA (Ambion Inc., Austin, TX) or snap frozen in liquid nitrogen (LN ). Lung samples were obtained from 25 consecutive perinatal autopsies of live-born and stillborn infants (median gestational age, 23 weeks) with various clinical presentations. Treatment of lung tissue with RNA yielded more total RNA and protein than LN freezing. The integrity of RNA, assessed by spectrophotometry and gel electrophoresis, was equivalent between both tissue preservation methods, and both methods produced RNA suitable for reverse transcriptase-polymerase chain reaction analysis of representative genes (beta-actin and surfactant protein-B [SP-B]). Similarly, the protein integrity of RNA -treated tissues was equivalent to that of LN -frozen tissues, as judged by Western blot analysis of SP-B/actin protein expression. Although the total yield was similar in live-born, nonmacerated stillborn and macerated stillborn infants, only RNA and protein from live-born or nonmacerated stillborn infants was suitable for subsequent molecular analyses. Within the 41-hour range studied, the duration of the postmortem interval did not affect the yield or integrity of RNA and protein with either tissue preservation method. In summary, high-quality RNA and protein, suitable for routine molecular analyses, can be obtained from postmortem lung tissue from live-born and nonmacerated stillborn infants, even with prolonged postmortem intervals. RNA is equivalent, if not superior, to LN for preservation of postmortem RNA and protein in developing human lungs.

Autolysis↗
PubMed2003

Applicability of different antibodies for the immunohistochemical localization of CFTR in respiratory and intestinal tissues of human and murine origin.

Cystic fibrosis (CF) is caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR) protein, which has a major role as a chloride (Cl(-)) channel. Although perhaps all functions of CFTR are still not fully characterized, localization studies are necessary to understand the consequences of the more than 1000 mutations thus far identified. Our aim was to determine the histological localization of CFTR on respiratory and colon epithelia of human and murine origin with a panel of several antibodies produced against different CFTR epitopes, using an indirect immunofluorescence method. Our results on human tissues confirm the apical localization of CFTR in ciliated cells of the respiratory mucosa and show that in colon tissue CFTR is observed in both apical and basolateral membranes of epithelial cells from colon crypts. However, poor tissue preservation of colon biopsies after immunohistochemistry (IHC) raises doubts about the latter localization. Contrary to human, mouse colon epithelium (not biopsed) presents good tissue preservation and evidences many cylindrical surface cells with high apical expression of CFTR. For the antibodies' sensitivity, we demonstrate that MATG1061, 24-1, M3A7, and MPCT-1 give good results, allowing the histological localization of CFTR protein of both human and murine origin.

Animals↗