Quantifying the calcification of abdominal aorta and major side branches with deep learning.

AIM: To explore the possibility of a neural network-based method for quantifying calcifications of the abdominal aorta and its branches. MATERIALS AND METHODS: In total, 58 computed tomography (CT) angiography volumes were selected from a dataset of 609 to represent different stages of sclerosis. The ground truth segmentations of the abdominal aorta, coeliac trunk, superior mesenteric artery, renal arteries, common iliac arteries, and their calcifications were delineated manually. Two V-Net ensemble models were trained, one for segmenting arteries of interest and another for calcifications. The branches of interest were shortened algorithmically. The volumes of calcification were then evaluated from the arteries of interest. RESULTS: The results indicate that automatic detection is possible with a high correlation to the ground truth. The scores for the ensemble calcification model were dice score of 0.69 and volumetric similarity (VS) of 0.80 and for the arteries of interest segmentations: aorta: dice 0.96, VS 0.98; aortic branches: dice 0.74, VS 0.87; and common iliac arteries: dice 0.72, VS 0.91. CONCLUSIONS: The presented neural network model is the first to be capable of automatically segmenting, in addition to calcification, both the aorta and its branches from contrast-enhanced CT angiography. This technology shows promise in addressing limitations inherent in earlier methods that relied solely on plain CT.

Prognostic Risk Factors for the Development of Compartment Syndrome in Acute Lower Limb Ischemia Patients Treated With Catheter-Directed Thrombolysis.

BACKGROUND: To determine predisposing factors that may lead to the development of compartment syndrome (CS) in patients with acute lower limb ischemia (ALLI) managed with intra-arterial catheter-directed thrombolysis (CDT). METHODS: This is a retrospective study of patients admitted between 01/2002 and 12/2015 to three university hospitals in Tampere, Turku, and Oulu, Finland, with acute or acute-on-chronic lower limb ischemia (Rutherford I-IIb). Patients managed with CDT and aspiration thrombectomies (AT) as an adjunct to CDT were included in the study. Multivariable binary logistic regression models were used to detect possible risk factors for the development of CS and its impact on the limb salvage and survival. Amputation-free survival (AFS) rates of CS and non-CS patients were compared using Kaplan-Meier survival analysis. The length of hospitalization was calculated and compared between the CS and non-CS groups. RESULTS: A total of 292 CDTs with or without ATs were performed on patients with a mean age of 71 years (standard deviation 13 years), 151 (51.7%) being male. Altogether, 12/292 (4.1%) treatment-related CS cases were registered. Renal insufficiency (odds ratio [OR] 4.27, P = 0.07) was associated with an increased risk of CS. All CS cases were managed with fasciotomies. Treatment with fasciotomy was associated with a prolonged hospitalization of a median of 7 days versus the 4 days for non-CS patients, P < 0.001. During the median follow-up of 51 months (interquartile range 72 months), 152/292 (52.1%) patients died and 51/292 (17.5%) underwent major amputations. CS was not associated with an increased risk of mortality, but it was associated with a higher risk of major amputation (OR 3.87, P = 0.027). The AFS rates of patients with or without CS did not significantly differ from each other in the long term. CONCLUSIONS: CS after CDT for the treatment of ALLI is uncommon. Renal insufficiency is associated with an increased risk of CS. Fasciotomy prolongs the hospitalization. Patients with CS are exposed to an increased risk of major amputation.

Toe Pressure and Toe Brachial Index are Predictive of Cardiovascular Mortality, Overall Mortality, and Amputation Free Survival in Patients with Peripheral Artery Disease.

OBJECTIVE/BACKGROUND: Peripheral haemodynamic parameters are used to assess the presence and severity of peripheral artery disease (PAD). The prognostic value of ankle brachial index (ABI) has been thoroughly delineated. Nonetheless, the relative usefulness of ankle pressure (AP), ABI, toe pressure (TP), and toe brachial index (TBI) in assessing patient outcome has not been investigated in a concurrent study setting. This study aimed to resolve the association of all four non-invasive haemodynamic parameters in clinically symptomatic patients with PAD with cardiovascular mortality, overall mortality, and amputation free survival (AFS). METHODS: In total, 732 symptomatic patients with PAD admitted to the Department of Vascular Surgery for conventional angiography at Turku University Hospital, Turku, Finland, between January 2009 and August 2011 were reviewed retrospectively. Demographic factors, cardiovascular mortality, all-cause mortality, and above foot level amputations were obtained and assessed in relation to AP, ABI, TP, and TBI by means of Kaplan-Meier life tables and a multivariate Cox regression model. RESULTS: The haemodynamic parameter that was associated with poor 36 month general outcome was TP < 30 mmHg. Univariate Cox regression analysis of stratified values showed that TP and TBI associated significantly with mortality. In multivariate analysis both TP and TBI were associated with a significant risk of death. For TP < 30 mmHg and TBI < 0.25 the risk of cardiovascular mortality was hazard ratio [HR] 2.84, 95% confidence interval [CI] 1.75-4.61 [p<.001]; HR 3.68, 95% CI 1.48-9.19 [p=.050], respectively; all-cause mortality (HR 2.05, 95% CI 1.44-2.92 [p<.001]; HR 2.53, 95% CI 1.35-4.74 [p=.040], respectively); and amputation or death (HR 2.13, 95% CI 1.52-2.98 [p<.001]; HR 2.46, 95% CI 1.38-4.40 [p=.050], respectively)... CONCLUSION: Among non-invasive haemodynamic measurements and pressure indices both TP and TBI appear to be associated with cardiovascular and overall mortality and AFS for patients with PAD presenting symptoms of the disease.

White matter damage of the brain is associated with poor outcome in vascular surgery patients with claudication: a pilot study.

OBJECTIVE: Peripheral arterial disease (PAD) is a systemic atherosclerotic syndrome with high post-operative morbidity and mortality. Fractional anisotropy (FA), an index measured by magnetic resonance diffusion tensor imaging (DTI), has been shown to be exceedingly sensitive to microstructural damage in brain white matter tracts. It is hypothesized that pre-operative white matter damage is more extensive in PAD patients scheduled for vascular surgery who experience an adverse long-term outcome. METHODS: Preoperative FA values were obtained in 24 consecutive PAD patients (age >40 years) scheduled for elective infrainguinal revascularization surgery and in 15 healthy age matched participants. All patients had their clinical history taken and underwent physical examination and laboratory tests. After surgery, patients were followed for a median of 52 months (range 40-63) and major adverse cardiovascular and cerebrovascular events (MACCE) were recorded. RESULTS: There were no statistically significant differences in baseline demographic or clinical variables between the MACCE group and the non-MACCE group. During follow up, eight PAD patients suffered a MACCE and they had lower FA values than patients without MACCE or healthy controls (mean ± SD 0.370 ± 0.017 vs. 0.392 ± 0.023 vs. 0.412 ± 0.018, p = .036 and p = .00007, respectively). Voxelwise analysis of the FA data revealed diffuse spatial distribution of white matter damage in PAD patients. There was no statistically significant association between the FA values and other clinical variables. CONCLUSION: Microstructural white matter damage was associated with poor outcome in PAD patients with claudication requiring surgical revascularization, and its extent may have clinical value in risk stratification.

Toe pressure and toe brachial index are predictive of cardiovascular mortality regardless of the most diseased arterial segment in symptomatic lower-extremity artery disease-A retrospective cohort study.

OBJECTIVE: Although lower extremity arterial disease (LEAD) is most often multisegmental, the predominant disease location and risk factors differ between patients. Ankle-brachial index (ABI), toe-brachial index (TBI), and toe pressure (TP) are predictive of outcome in LEAD patients. Previously, we reported a classification method defining the most diseased arterial segment (MDAS); crural (CR), femoropopliteal (FP), or aortoiliac (AOI). Current study aimed to analyze the associations between MDAS, peripheral pressure measurements and cardiovascular mortality. MATERIALS AND METHODS: We reviewed retrospectively 729 consecutive LEAD patients (Rutherford 2-6) who underwent digital subtraction angiography between January, 2009 to August, 2011 and had standardized peripheral pressure measurements. RESULTS: In Cox Regression analyses, cardiovascular mortality was associated with MDAS and non-invasive pressure indices as follows; MDAS AOI, TP <30 mmHg (HR 3.00, 95% CI 1.13-7.99); MDAS FP, TP <30 mmHg (HR 2.31, 95% CI 1.36-3.94), TBI <0.25 (HR 3.20, 95% CI 1.34-7.63), ABI <0.25 (HR 5.45, 95% CI 1.56-19.0) and ≥1.30 (HR 6.71, 95% CI 1.89-23.8), and MDAS CR, TP <30 mmHg (HR 4.26, 95% CI 2.19-8.27), TBI <0.25 (HR 7.71, 95% CI 1.86-32.9), and ABI <0.25 (HR 2.59, 95% CI 1.15-5.85). CONCLUSIONS: Symptomatic LEAD appears to be multisegmental with severe infrapopliteal involvement. Because of this, TP and TBI are strongly predictive of cardiovascular mortality and they should be routinely measured despite the predominant disease location or clinical presentation.

Expression of beta-nerve growth factor and its receptor in rat seminiferous epithelium: specific function at the onset of meiosis.

beta-Nerve growth factor (NGF) is expressed in spermatogenic cells and has testosterone-downregulated low-affinity receptors on Sertoli cells suggesting a paracrine role in the regulation of spermatogenesis. An analysis of the stage-specific expression of NGF and its low affinity receptor during the cycle of the seminiferous epithelium in the rat revealed NGF mRNA and protein at all stages of the cycle. Tyrosine kinase receptor (trk) mRNA encoding an essential component of the high-affinity NGF receptor was also present at all stages. In contrast, expression of low affinity NGF receptor mRNA was only found in stages VIIcd and VIII of the cycle, the sites of onset of meiosis. The low-affinity NGF receptor protein was present in the plasma membrane of the apical Sertoli cell processes as well as in the basal plasma membrane of these cells at stages VIIcd to XI. NGF was shown to stimulate in vitro DNA synthesis of seminiferous tubule segments with preleptotene spermatocytes at the onset of meiosis while other segments remained nonresponsive. We conclude that NGF is a meiotic growth factor that acts through Sertoli cells.

Pattern of orexin expression and direct biological actions of orexin-a in rat testis.

Orexins, hypothalamic neuropeptides initially involved in the control of food intake and sleep-wake cycle, have recently emerged as pleiotropic regulators of different biological systems, including the reproductive axis. Besides central actions, peripheral expression and functions of orexins have been reported, and prepro-orexin and orexin type-1 receptor mRNAs have been detected in the testis. However, the pattern of expression and biological actions of orexin in the male gonad remain mostly unexplored. In this study, we report analyses on testicular prepro-orexin mRNA expression and orexin-A immunoreactivity in different experimental settings, and on direct effects of orexin-A on seminiferous tubule functions. Expression of prepro-orexin mRNA was demonstrated in the rat testis at different stages of postnatal development, with negligible levels at early juvenile period and maximum values in adulthood. Likewise, orexin-A immunoreactivity was demonstrated along postnatal maturation, with strong peptide signal in Leydig cells and spermatocytes at specific stages of meiosis. Testicular expression of prepro-orexin mRNA appeared hormonally regulated; its levels decreased after hypophysectomy and increased after gonadotropin replacement and ghrelin stimulation. Finally, orexin-A suppressed the expression of key Sertoli cell genes, such as Müllerian-inhibiting substance and stem cell factor, and inhibited DNA synthesis in specific stages of the seminiferous epithelium. In conclusion, we provide evidence for the regulated expression of orexin in the rat testis and its potential involvement in the control of seminiferous tubule functions. Together with our recent results on the expression of orexin type-1 receptor in the rat testis, our data further document a novel testicular site of action of orexins in the control of male reproductive axis.

Polyamines and regulation of spermatogenesis: selective stimulation of late spermatogonia in transgenic mice overexpressing the human ornithine decarboxylase gene.

Polyamines are believed to participate in the induction of cell growth, differentiation, and proliferation, but their role in spermatogenesis has remained obscure. Two transgenic mouse lines (K2 and K15) that overexpress the human ornithine decarboxylase (ODC) gene coding for a rate-controlling enzyme in polyamine biosynthesis and, hence, contain high levels of tissue putrescine have been used to study the stage-specific role of ODC in spermatogenesis. In K2 mice with 30-fold testicular ODC overexpression, [3H]thymidine incorporation at stages I-VI of the cycle of the seminiferous epithelium was significantly above the control level. This may reflect a specific stimulation of DNA synthesis in type A4, intermediate, and type B spermatogonia. The K15 mice that have about 70-fold ODC overexpression showed an elevation of DNA synthesis only at stage V of the cycle, suggesting a specific dependence of type B spermatogonia on putrescine. In K15 mice, [3H]thymidine incorporation of stage VIII tubule segments was decreased, suggesting that excess amounts of putrescine selectively inhibit meiotic DNA synthesis. We propose that putrescine has strictly selective local stimulatory and inhibitory actions during spermatogenic DNA synthesis, and that its excess amounts ultimately may lead to decreased fertility.

Testosterone inhibits and induces apoptosis in rat seminiferous tubules in a stage-specific manner: in situ quantification in squash preparations after administration of ethane dimethane sulfonate.

The quantitative effects of ethane dimethane sulfonate (EDS), a Leydig cell toxin, on apoptosis in adult rat seminiferous epithelium were studied by the improved transillumination method. Nonradioactive in situ end labeling of fragmented DNA in squash preparations revealed significant increases in apoptotic cells in stages II-XI, whereas controls showed 0.5-2.3 apoptotic cells/mm tubule. Seven days post-EDS treatment, the highest numbers of apoptotic cells were scored in stages VIIab and VIIcd (74.7 +/- 23.8 and 61.3 +/- 16.0 cells/mm, respectively). The effects were suppressed by testosterone (T) supplementation, except in stages II-III and VIIcd. An opposite effect was found in stage XII, where the number of apoptotic cells decreased 1, 3, and 7 days after EDS treatment and returned to control levels in T-supplemented rats. Electrophoretic analysis of internucleosomal DNA fragmentation revealed a biphasic apoptotic process after 1 and 5-7 days due to Leydig and germ cell apoptosis, respectively. The specific germ cell apoptosis was also confirmed by electron microscopic analysis. The results suggest that T withdrawal induces apoptotic cell death in most stages of the cycle and that the effects are largely preventable. In stage XII, however, T seems to promote apoptosis in premeiotic cells.

Effects of activin-A, inhibin-A, and transforming growth factor-beta 1 on stage-specific deoxyribonucleic acid synthesis during rat seminiferous epithelial cycle.

Activin and inhibin are members of the transforming growth factor-beta (TGF beta) gene family. They are expressed in various organ systems, where they possess regulatory functions. Inhibin, activin, and TGF beta have been reported to also be expressed in the adult rat testis. We studied in vitro the action of these growth factors on premitotic and premeiotic DNA synthesis during the rat seminiferous epithelial cycle. Two-millimeter rat seminiferous tubule segments were isolated by transillumination-assisted microdissection from stages V, VIIa, VIII-IX, and I of the cycle and incubated in vitro in the presence of activin-A, inhibin-A, or TGF beta 1. During 24-, 48-, and 72-h incubation spontaneous progression of spermatogenesis was noted. The staged samples allowed us to selectively quantitate DNA synthetic activity of specific germ cell types. At the end of the culture, the tubules were pulse labeled with [3H]thymidine, and DNA synthesis was quantified by liquid scintillation counting, and the activated cells were detected by autoradiography. Activin-A stimulated preleptotene spermatocyte DNA synthesis in a dose-dependent manner. DNA synthesis of intermediate spermatogonia was also stimulated by activin-A, whereas inhibin-A inhibited DNA synthesis of these cells. TGF beta 1 had a small, but significant, stimulatory effect on DNA synthetic activity at stage VII. These results support the view that activin-A, inhibin-A, and TGF beta 1 take part in the regulation of DNA synthesis during rat spermatogenesis.

In vitro, follicle-stimulating hormone prevents apoptosis and stimulates deoxyribonucleic acid synthesis in the rat seminiferous epithelium in a stage-specific fashion.

The effects of FSH on stage-specific apoptosis and DNA synthesis in the adult rat seminiferous epithelium were studied in vitro. Seminiferous tubular segments from stages I, V, VIIa, and VIII-IX were cultured for 24, 48, and 72 h in different concentrations of FSH. Apoptotic cells were detected by in situ end labeling of DNA strands and quantified from squash preparations. After 48 h of culture, a FSH concentration of 2 ng/ml prevented apoptosis of early (steps 1-3) spermatids. In stage VIII-IX tubules cultured for 72 h, FSH decreased the apoptosis of pachytene spermatocytes. An apoptotic type of cell death of germ cells was confirmed by DNA laddering, electron microscopy, supravital acridine orange staining, and phase contrast microscopy of unstained living cells. The effects of FSH on stage-specific DNA synthesis were studied using the same culture system. FSH increased [3H]thymidine incorporation specifically at stages I and VIII-IX, and autoradiography confirmed stimulation of mitotic and meiotic DNA synthesis in type B spermatogonia and preleptotene spermatocytes, respectively. Increased thymidine incorporation also suggested that FSH stimulated DNA synthesis of type A and intermediate spermatogonia. Most effects exerted by FSH were seen in stages containing high levels of FSH receptors and FSH-stimulated cAMP production. In conclusion, the results suggest that FSH, probably acting via Sertoli cells, has a regulatory function in spermatogenic apoptosis and DNA synthesis in stages previously demonstrated to be preferentially dependent on FSH stimulation.

Function of stem cell factor as a survival factor of spermatogonia and localization of messenger ribonucleic acid in the rat seminiferous epithelium.

To address the possibility that stem cell factor (SCF) is a paracrine regulator of germ cell development in the adult rat testis, stage-specific distribution of SCF messenger RNA (mRNA) was investigated with Northern blot and in situ hybridization analyses. The highest levels of SCF mRNA were found in stages II-VI of the rat seminiferous epithelial cycle, whereas the lowest levels were in stages VII-VIII. Intermediate levels of SCF mRNA were detected in stages IX-XIV-I of the cycle. The expression of the SCF gene was found to be developmentally regulated, and the expression pattern followed the process of Sertoli cell proliferation and differentiation during postnatal life. The effect of mouse recombinant SCF on spermatogonial DNA synthesis was studied using an in vitro tissue culture system for stage-defined seminiferous tubules. A significant increase in DNA synthesis in spermatogonia could be detected when tubule segments from stage XII were cultured in the presence of 100 ng/ml SCF for 48 h (P < 0.05) and 72 h (P < 0.01). This observation was further confirmed with autoradiographic analyses; almost a 100-fold increase in thymidine incorporation in the SCF-treated (100 ng/ml) tubule segments was observed compared with that in untreated samples. The results of the present study suggest that SCF is a Sertoli cell-produced paracrine regulator and acts as a survival factor for spermatogonia in the adult rat seminiferous epithelium in a stage-specific manner.

Function of interleukin-6 as an inhibitor of meiotic DNA synthesis in the rat seminiferous epithelium.

Interleukin-6 bioactivity (IL-6) has been shown to be present in Sertoli cells. To further characterize the IL-6 in the seminiferous epithelium, the IL-6 like-antigen was detected, stage-specific basal distribution of IL-6-like bioactivity and its regulation by FSH, cAMP and TPA was characterized in isolated, rat seminiferous tubule segments. In addition, the effects of human recombinant IL-6 on stage-specific DNA synthesis was investigated. Both monoclonal and polyclonal antibodies recognized M(r) 22 and 23 kDa of IL-6 like immunoreactivity in the seminiferous epithelium. The basal IL-6 production showed high levels during stages XIII-XIV-I-V, low during VII and VIII. FSH stimulated IL-6 production at nearly all stages and most significantly at stage VII of the cycle. Human recombinant IL-6 dose-dependently inhibited the onset of meiotic DNA synthesis of preleptotene spermatocytes, and a minor inhibition was found on advanced (A3-type B) spermatogonia. These results support the hypothesis that IL-6 is a stage-specific paracrine regulator of the seminiferous epithelium exerting a specific inhibitory action on meiotic DNA synthesis.

MIP-1 alpha is a regulator of mitotic and meiotic DNA synthesis during spermatogenesis.

To find out whether macrophage inflammatory protein-1 alpha (MIP-1 alpha) has a role in the regulation of germ cell development, we studied its effects on spermatogenic stage-specific DNA synthesis in vitro. MIP-1 alpha increased the DNA synthesis of primitive type A2-4 spermatogonia and of premeiotic cells, whereas the DNA synthesis of more differentiated intermediate and type B spermatogonia was inhibited when cultured in the presence of MIP-1 alpha. An antibody against MIP-1 alpha cross-reacted with a protein of 15 kDa from every spermatogenic stage of rat seminiferous epithelium. Immunohistochemical studies with the same antibody revealed a complex pattern of MIP-1 alpha localization both in primitive and advanced spermatogenic cells. These observations suggest that MIP-1 alpha is a local regulator of mitotic and meiotic DNA synthesis.

Follicle-stimulating hormone regulates the expression of cyclic protein-2/cathepsin L messenger ribonucleic acid in rat Sertoli cells in a stage-specific manner.

Cyclic protein-2/cathepsin L (CP-2) is secreted by Sertoli cells in a highly stage-specific manner, maximally during stages VI-VII of the rat seminiferous epithelial cycle. We investigated FSH regulation of CP-2 mRNA expression of its cellular localization in isolated staged seminiferous tubular segments. FSH induced a significant increase of CP-2 mRNA expression and its cellular localization in isolated staged seminiferous tubular segments. FSH induced a significant increase of CP-2 mRNA levels in stages IX-I, whereas in stages II-VIII, the levels of CP-2 mRNA were reduced. A similar effect was produced by two cAMP analogs, dbcAMP (0.2 mM) and Sp cAMP (20 microM). FSH and cAMP did not affect on the levels of SGP-2 mRNA during the seminiferous epithelial cycle. The magnitude of the response was time- and dose-dependent; the maximum was obtained with 100 ng/ml of FSH. It is likely that FSH regulates Cp-2 gene transcription, since de novo RNA synthesis was required for the stimulatory FSH effect on CP-2 mRNA levels, while ongoing protein synthesis was not. In conclusion, the data suggest that FSH, via cAMP-mediated pathway, regulates CP-2/cathepsin L gene transcription in rat Sertoli cells and modulated the stage-specific expression pattern.

Immunosuppressive activity in the rat seminiferous tubules.

Rat seminiferous tubule segments in defined stages of the epithelial cycle were isolated by transillumination-assisted microdissection. The segments were cultured together with ConA-stimulated peripheral blood lymphocytes (PBL) and incorporation of 3H-labelled thymidine was measured. Tubule segments in stages II-VIII of the seminiferous epithelial cycle inhibited PBL proliferation significantly more than stages IX-I. Inhibition was lowest in stages IX-XII and increased progressively to reach a maximum in stages II-VIII. In a more detailed analysis, tubules in stages V and VI inhibited PBL proliferation significantly less than stage II tubules. No significant difference was observed between stages II and VII. The immunosuppressive activity had molecular weights of approximately 25 kDa and approximately 65 kDa in stage II-VIII seminiferous tubules. In stage II-VI seminiferous tubules activity was present also at approximately 10 kDa. The results suggest that the seminiferous tubules produce high-molecular weight immunosuppressive activity in a stage-dependent way. In addition to its contribution to the immunologically privileged milieu of the testis this activity may also be involved in the physiological regulation of DNA synthesis in the seminiferous epithelium.

Effects of etoposide on stage-specific DNA synthesis during rat spermatogenesis.

The stage-specific effect of etoposide on spermatogenic DNA synthesis was measured 1, 3 and 18 days after a single intraperitoneal injection of etoposide. Etoposide inhibited premitotic DNA synthesis most effectively at stages II-III and IV-V of the seminiferous epithelial cycle in which DNA synthesis of late spermatogonia takes place. Compared with control levels, DNA synthesis at stages II-III was maximally inhibited 43% and 57% at doses of 5 and 10 mg/kg, respectively, and at stages IV-V the maximal inhibition was 67% and 62%, at doses of 5 and 10 mg/kg respectively. Premeiotic DNA synthesis was not as vulnerable to the etoposide action as premitotic DNA synthesis, the maximal inhibition of premeiotic DNA synthesis was 39% and 41% compared with control at doses of 5 and 10 mg/kg, respectively. Induction of most probably repair-type DNA synthesis was demonstrated in stages I-III, VIIa-b and XII of the cycle. All the effects of etoposide were most apparent 1 and 3 days after treatment but had not totally disappeared 18 days after the treatment.

Mice with a deletion in the first intron of the Col1a1 gene develop dissection and rupture of aorta in the absence of aneurysms: high-resolution magnetic resonance imaging, at 4.7 T, of the aorta and cerebral arteries.

Deletion of the majority of the first intron of the Col1a1 gene in mice leads to decreased type I collagen synthesis and content in the aortic wall. In 54% of cases, mice homozygous for the Col1a1 mutation die of thoracic hemorrhage by the age of 18 months. It is unknown whether the fatal bleeding results from an acute dissection of the aortic wall or a gradually developing dilatation of the medial layer prior to rupture. We optimized high-resolution MRI methods using a 4.7 T MR scanner to obtain in vivo images of the entire mouse aorta. The MR images were acquired in three imaging planes using gradient echo, spin echo, and spin echo with inversion recovery pulse sequences with a maximum in-plane resolution of 68 x 68 microm and acquisition times less than 10 min. In five Col1a1 mutated mice aged 16 months, the MR images showed no signs of aneurysmal dilatation, wall defects, or former dissection, suggesting that the mechanism for aortic rupture is an acute dissection of the aortic medial layer. Cerebral arteries were imaged using a three-dimensional time of fight pulse sequence. The resolution of 73 x 73 x 94 microm showed normal cerebral arteries. Histology showed a 22% thinner cerebral artery wall in Col1a1 mutated mice.

In-situ quantification of stage-specific apoptosis in the rat seminiferous epithelium: effects of short-term experimental cryptorchidism.

Two techniques have been combined for quantification of apoptotic germ cells in defined stages of the cycle of the seminiferous epithelium: the improved transillumination method and nonradioactive in-situ end-labelling of DNA (ISEL). Segments of rat seminiferous tubules were squashed between a microscope slide and coverslip, and the stage identified under a phase-contrast microscope. After fixation, apoptotic cells were detected by ISEL and scored per 1 mm tubule. In the normal testis apoptotic cells were found in all stages, the highest frequency occurring in stages XII-XIV (19 cells/mm). In short-term (24 and 48 h) experimentally cryptorchid testes, a significant increase in number of apoptotic germ cells was evident in all stages, except for VI and VIII. Apoptosis of germ cells was confirmed by electrophoresis of radioactively labelled DNA from stages VII-VIII and XIII-I. It is proposed that apoptosis is a means of eliminating the most sensitive germ cells after short-term experimental cryptorchidism.

Expression of mitochondrial heat shock protein 60 in distinct cell types and defined stages of rat seminiferous epithelium.

Changes in the level of the gene transcript of heat shock protein (hsp)60, a mitochondrial chaperonin, during the cycle of rat seminiferous epithelium and its cellular localization were studied. The seminiferous epithelium showed a cell type-specific expression of hsp60. Immunostaining of adult rat testis revealed localization in Sertoli and Leydig cells. In germ cells, mitochondria of spermatogonia and early primary spermatocytes were immunoreactive for hsp60. Mitochondria of all other germ cell types were completely negative for hsp60. Stage-specific expression of hsp60 was determined from pooled segments of stage-specific microdissected tubules by a combination of Western blotting and polymerase chain reaction (PCR). High concentrations of hsp60 were found in stages I-V and IX-XIV, and low levels were detected in the other stages, i.e., VI-VIII. In stages with high hsp60 expression, spermatogonia divide mitotically, whereas in stages lacking mitosis, the hsp60 level was much weaker. In seminiferous epithelium, two different types of mitochondria are present. Therefore, immunoelectron microscopy was used to differentiate these two morphologically distinct types of mitochondria. The crista type of mitochondria (e.g., in Sertoli cells and spermatogonia) reacted with the antibody against hsp60, whereas hsp60 was negative in so-called "condensed"-type mitochondria found in midpachytene spermatocytes and more advanced germ cells. It could be shown for the first time that expression of the hsp60 gene is regulated during the cycle of the seminiferous epithelium. The results indicate that the gene product is primarily needed during the initial steps of spermatogenesis in which most of the cell divisions occur, while its expression during the differentiation of spermatids and sperm is obviously not necessary. The presence of hsp60 in stages with mitotic activity suggests a very active mitochondrial protein import and protein assembly machinery that generates further mitochondria for the dividing cells.