Collagenase predigestion on paraffin sections enhances collagen immunohistochemical detection without distorting tissue morphology

Reliable immunohistochemical detection of collagen in formalin fixed, paraffin embedded tissues requires protease digestion. While these pan-proteases (pepsin, trypsin, protease K, etc.) enhance collagen detection, they also digest many other tissue proteins and produce poor cellular morphology and unrecognizable cellular structures. Balancing the conditions (protease type, concentration, incubation time and temperature) to digest some, but not all, proteins in a tissue section while optimizing collagen detection requires one to compromise improved collagen immunolabeling with adequate cellular morphology. Furthermore, optimal conditions for digesting tissue proteins to enhance collagen detection vary among tissue types and their fixation. Although brain is not typically subject to these deleterious consequences, structures such as epithelium, spermatids, stroma etc. and other tissues with complicated histology are profoundly affected. To resolve this technical dilemma, we discovered a novel use for collagenase to enhance collagen immunodetection without affecting the noncollagen proteins, thereby preserving tissue morphology. Collagenase, which is typically used in vitro for disassociation of cells, has never been used reliably on formalin fixed, paraffin embedded tissue sections. This new use of collagenase for immunohistochemistry promotes increased collagen immunolabeling, is easy to use, is versatile, and allows preservation of tissue structure that provides maximal and accurate histological information.     

Expression of estrogen receptors alpha and beta in the corpus luteum and uterus from non-pregnant and pregnant llamas

Because estrogen may be involved in maternal recognition of pregnancy and embryonic migration in llamas, expression of estrogen receptor subtypes alpha (ERalpha) and beta (ERbeta) was evaluated in corpus luteum (CL), endometrium, and uterus using relative RT-PCR. Tissues were recovered from sterile-mated (SM) and pregnant (PG) females during Days 7-11 and 7-13 (Day 0 = day of mating), respectively, and follicular phase and juvenile females. Luteal expression of ERalpha and beta was similar (P > 0.10) in SM and PG females and within Days 7-11, however, expression of ERalpha in ovarian tissue from follicular phase females was greater (P < 0.05) than Days 7 and 9 CL. Uterus expressed less ERalpha and beta compared to endometrium (P = 0.07 and P < 0.01, respectively). Expression of ERalpha was greater (P < 0.05) in Day 7 and follicular phase uteri than Days 9 and 11, Day 13 PG and juvenile uteri. Uterine ERbeta expression was greater (P = 0.09) in PG versus SM females and in mated compared to follicular phase females (P < 0.05). Endometrial expression of ERalpha and beta did not differ (P > 0.10) between SM and PG females or by day. The presence of luteal ER during this period may mean a role for estradiol in maternal recognition of pregnancy. Observed increases in uterine ER expression with no changes in endometrium suggest expression increased in myometrium and/or perimetrium. Upregulation of myometrial ERbeta in PG females may be involved in supporting uterine migration of the embryo.     

Investigation of apoptosis in cultured cells infected with equine herpesvirus 1

Many viruses alter different stages of apoptosis of infected cells as a strategy for successful infection. Few studies have addressed mechanisms of equine herpesvirus 1 (EHV-1) strain-induced cell death. We investigated the effect of an abortigenic strain (AR8 strain) on heterologous Madin–Darby bovine kidney cells and homologous equine dermis (ED) cells cell lines. We compared morphologic and biochemical features of early and late apoptosis at different postinfection times. We investigated translocation of phosphatidylserine to the cell surface, nuclear fragmentation and changes in the cytoskeleton using flow cytometry and annexin V/propidium iodide staining, DNA laddering, terminal deoxynucleotidyl transferase UTP nick-end labeling assay and immunofluorescence staining of cytokeratin 18 cleavage. AR8 EVH-1 strain interfered with apoptosis in both cell lines, particularly during the middle stage of the replication cycle; this was more evident in ED cells. Although this antiapoptotic effect has been reported for other alpha herpesviruses, our findings may help elucidate how EHV-1 improves its infectivity during its cycle.     

Stent implantation through a self-expanding stent

Jailing of a side-branch is a known complication of stent implantation, and makes access to the side-branch difficult, especially if the stent is of the self-expanding type. Although plain balloon angioplasty is feasible for the jailed side-branches, the use of newer devices (a stent, Rotablation or atherectomy) has not been described. We describe a novel way of treating a side-branch jailed by a self-expanding stent by using stent implantation through the strut of a self-expanding stent.     

Development of mouse embryos in media containing lectins

Lectins known to stimulate mitosis in cultured cells were evaluated for effects on development of mouse embryos in vitro. Two-cell mouse embryos were cultured in one of the following treatments: Whitten's medium as the control medium; Whitten's medium with 1, 10 or 100 mug/ml concanavalin A; Whitten's medium with 1, 10 or 100 mug/ml leucoagglutinin; Whitten's medium with 1, 10 or 100 mug/ml phytohemagglutinin; Whitten's medium with 1, 10 or 100 mug/ml pokeweed-mitogen; and Whitten's medium with 1, 10 or 100 mug/ml wheat germ agglutinin. Development to the morula stage was blocked in media with 100 mug/ml concanavalin A and 10 and 100 mug/ml wheat germ agglutinin, whereas blastocyst formation was blocked in all pokeweed-mitogen supplemented media. Embryos incubated in 10 and 100 mug/ml wheat germ agglutinin underwent premature cavitation or vacuolation at 24 to 48 h of culture. More embryos formed blastocysts in media with 1 and 100 mug/ml phytohemagglutinin and 10 mug/ml leucoagglutinin than in Whitten's medium (P<0.05). The percentage of embryos hatching was greatest in 1 mug/ml phytohemagglutinin (P<0.05), but it was the same in Whitten's medium, 1 mug/ml concanavalin A and 1 mug/ml leucoagglutinin (P>0.05). Cell division was not stimulated by the lectins; however, it was significantly suppressed in media with 10 and 100 mug/ml concanavalin A, 100 mug/ml phytohemagglutinin, 1, 10 and 100 mug/ml pokeweed-mitogen, and 10 and 100 mug/ml wheat germ agglutinin. Solubility of the zona pellucida in sodium isothicyanate (NaSCN) was reduced in 100 mug/ml phytohemagglutinin, 100 mug/ml leucoagglutinin and 1 mug/ml wheat germ agglutinin media (P<0.05) when compared to Whitten's medium and may have accounted for the reduced hatching observed in these treatments. Development of isolated blastomeres into blastocysts was reduced in media with 1 mug/ml wheat germ agglutinin, 1 mug/ml concanavalin A, and 10 and 100 mug/ml leucoagglutinin (P<0.05) but was similar in media with 1 mug/ml leucoagglutinin and 1, 10 and 100 mug/ml phytohemagglutinin when compared to Whitten's medium (P>0.05). The extent of embryo development in media with lectins depended upon the degree of cytotoxicity and potential biochemical modifications induced in the zona pellucida. Greatest embryo development took place in medium with 1 mug/ml phytohemagglutinin; however, the mechanism was not that of stimulation of cell division or a change in zona pellucida solubility.     

Protein content of porcine embryos during the first nine days of development

Little is known about the physiochemical aspects of porcine embryos prior to implantation. The purpose of this study was to quantitate the total protein content of porcine embryos from fertilization through day 9 of development. Thirty-seven gilts and two sows were hand mated and the reproductive tracts collected at slaughter 1 to 9 days after the onset of estrus. The uteri were flushed with phosphate buffered saline (PBS), and embryos were collected, washed with PBS and transferred directly to test tubes containing distilled water. Only embryos which appeared morphologically normal were used. Protein content was determined by the Bio-Rad microassay. Standard curves were constructed for each assay using. 8 to 19 mug gamma globulin (Bio-Rad assay). Protein standards were run in triplicate. Regression lines were calculated for protein standards, and the resulting line was used to determine total protein in the unknown samples. Protein content of unfertilized eggs and embryos increased steadily through days 3, 4 and 5 of development, from 273 to 334, 491 and 620 ng, respectively. This result suggests that the protein content of porcine embryos increases only slightly from fertilization through day 5 of development. A dramatic increase in embryo protein content was observed between days 6 and 9 of development, which is the time of blastocyst formation and hatching of the embryo from the zona pellucida.     

Occurrence of a non-cellular suprazonary layer in embryos collected from prepuberal calves

Twenty Angus calves between 4 and 7 months of age were randomly assigned to one of two superovulation treatment groups. Group I consisted of ten calves which were injected intramuscularly with 50 mg of progesterone 4 and 2 days before injection with 1200 I.U. of PMSG followed 72 hrs later by 50 mg of LH given intravenously. Group II consisted of ten calves which were not injected with progesterone before receiving PMSG and LH as in Group I. Both groups of calves were inseminated by the rectal fixation method with two straws of frozen semen 72 hrs after PMSG injection and at subsequent 12 hr intervals for a total of four inseminations. All semen was extended from a single ejaculate from one bull. Embryos were recovered by surgery or slaughter 48 to 72 hrs following the last insemination. A total of 80 and 70 ovulations were recorded from treatment Group I and II, respectively. Recovery and fertilization rates were 66 and 57% following progesterone treatment and 67 and 51% in calves without progesterone pretreatment. Seventy-seven percent ($\text{23}\text{30}$) of the fertilized embryos recovered in treatment Group I exhibited a suprazonary layer. This suprazonary layer appeared to be non-cellular on the basis of eosin-hematoxylin staining and ranged in thickness from 3 to 24 μm. All fertilized embryos in treatment Group II and unfertilized eggs in treatment Groups I and II, possessed completely smooth zonae pellucidae with no evidence of a suprazonary layer. These observations suggest that the conditions of the progesterone treated prepuberal tract, coupled with the process of sperm penetration of the oocyte, result in the formation of a non-cellular layer which surrounds the zona pellucida to varying degrees of thickness. The influence of this suprazonary layer on embryo viability in prepuberal calves remains to be determined.     

EDTA chelation therapy for cardiovascular disease: a systematic review

Background

Experimental studies support an important role for endothelial nitric oxide synthase (eNOS) in the regulation of angiogenesis. In humans, a common polymorphism exists in the eNOS gene that results in the conversion of glutamate to aspartate for codon 298. In vitro and in vivo studies have suggested a decreased NOS activity in patients with the Asp²⁹⁸ variant. We hypothesized that a genetic-mediated decreased eNOS activity may limit collateral development in patients with chronic coronary occlusions.

Methods

We selected 291 consecutive patients who underwent coronary angiography and who had at least one chronic (>15 days) total coronary occlusion. Collateral development was graded angiographically using two different methods: the collateral flow grade and the recipient filling grade. Genomic DNA was extracted from white blood cells and genotyping was performed using previously published techniques.

Results

Collateral development was lower in patients carrying the Asp²⁹⁸ variant than in Glu-Glu homozygotes (collateral flow grade: 2.64 ± 0.08 and 2.89 ± 0.08, respectively, p = 0.04; recipient filling grade: 3.00 ± 0.08 and 3.24 ± 0.07, respectively, p = 0.04). By multivariable analysis, three variables were independently associated with the collateral flow grade: female gender, smoking, and the Asp²⁹⁸ variant (p = 0.03) while the Asp²⁹⁸ variant was the sole variable independently associated with the recipient filling grade (p = 0.03).

Conclusion

Collateral development is lower in patients with the Asp²⁹⁸ variant. This may be explained by the decreased NOS activity in patients with the Asp²⁹⁸ variant. Further studies will have to determine whether increasing eNOS activity in humans is associated with coronary collateral development.