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Understanding and treatment of spinal cord pathology is limited in part by a lack of longitudinal in vivo imaging strategies at the cellular level. We developed a chronically implanted spinal chamber and surgical procedure suitable for time-lapse in vivo multiphoton microscopy of mouse spinal cord without the need for repeat surgical procedures. Repeated imaging was routinely achieved for more than five weeks post-operatively with up to ten separate imaging sessions. We observed neither motor function deficit nor neuropathology in the spinal cord as a result of chamber implantation. Using this chamber we quantified microglia and afferent axon dynamics following a laser-induced spinal cord lesion and observed massive microglia infiltration within one day along with a heterogeneous dieback of axon stumps. By enabling chronic imaging studies over timescales ranging from minutes to months, our method offers an ideal platform for understanding cellular dynamics in response to injury and therapeutic interventions.

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Abstract. Minute virus of canines (MVC, canine parvovirus type-1) caused inapparent to severe illness in neonatal specific-pathogen-free pups exposed by the oronasal route. The experimental disease was generally mild. Four of 21 infected pups had clinical signs of respiratory illness, but only 2 pups, not euthanized during the early postinoculation period, developed severe illness or died. Principal pathologic changes included bronchitis and interstitial pneumonia with various degrees of lymphadenitis. In contrast to the reported field cases, enteric signs were absent in the experimentally infected animals. Histopathologic changes in the small intestine were mild or absent. Bronchial, bronchiolar, and alveolar epithelial cells appeared to be the sites of initial and most extensive viral growth, reflecting the pattern of histopathologic changes. The disease caused by MVC was mild in comparison to that caused by canine parvovirus-type 2. MVC now appears to be established as a cause of illness in young pups and of transplacental infections with embryo resorption. The prevalence of MVC hemagglutination-inhibiting antibodies was high (~50%) in adult dog sera from widely separated geographic areas of the United States.

The RAD9-RAD1-HUS1 (9-1-1) complex is a heterotrimeric PCNA-like clamp that responds to DNA damage in somatic cells by promoting DNA repair as well as ATR-dependent DNA damage checkpoint signaling. In yeast, worms, and flies, the 9-1-1 complex is also required for meiotic checkpoint function and efficient completion of meiotic recombination; however, since Rad9, Rad1, and Hus1 are essential genes in mammals, little is known about their functions in mammalian germ cells. In this study, we assessed the meiotic functions of 9-1-1 by analyzing mice with germ cell-specific deletion of Hus1 as well as by examining the localization of RAD9 and RAD1 on meiotic chromosomes during prophase I. Hus1 loss in testicular germ cells resulted in meiotic defects, germ cell depletion, and severely compromised fertility. Hus1-deficient primary spermatocytes exhibited persistent autosomal γH2AX and RAD51 staining indicative of unrepaired meiotic DSBs, synapsis defects, an extended XY body domain often encompassing partial or whole autosomes, and an increase in structural chromosome abnormalities such as end-to-end X chromosome-autosome fusions and ruptures in the synaptonemal complex. Most of these aberrations persisted in diplotene-stage spermatocytes. Consistent with a role for the 9-1-1 complex in meiotic DSB repair, RAD9 localized to punctate, RAD51-containing foci on meiotic chromosomes in a Hus1-dependent manner. Interestingly, RAD1 had a broader distribution that only partially overlapped with RAD9, and localization of both RAD1 and the ATR activator TOPBP1 to the XY body and to unsynapsed autosomes was intact in Hus1 conditional knockouts. We conclude that mammalian HUS1 acts as a component of the canonical 9-1-1 complex during meiotic prophase I to promote DSB repair and further propose that RAD1 and TOPBP1 respond to unsynapsed chromatin through an alternative mechanism that does not require RAD9 or HUS1.

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Early embryonic losses are much higher in nuclear transfer (cloned) pregnancies, and this is a major impediment to improving the efficiency of cloned animal production. In cattle, many of these losses occur around the time of placental attachment from the fourth week of gestation. We studied the potential for altered immunologic status of cloned pregnancies to be a contributing factor to these embryonic losses. Expression of major histocompatibility complex class I (MHC-I) by trophoblast cells and distribution of endometrial T-lymphocyte numbers were investigated. Six 5-wk-old cloned pregnancies were generated, and 2 others at 7 and 9 wk were also included, all derived from the same fetal cell line. All 8 cloned placentas displayed trophoblast MHC-I expression. None of the 8 controls (4-7 wk old) showed any MHC-I expression. The percentage of trophoblast cells expressing MHC-I varied in the clones from 17.9% to 56.5%. Numbers of T lymphocytes (CD3(+) lymphocytes) were significantly higher in the endometrium of the majority of cloned pregnancies compared with controls. In the cloned pregnancies, large aggregates of T cells were frequently observed in the endometrium in addition to increased numbers of diffusely spread subepithelial lymphocytes. As trophoblast MHC-I expression is normally suppressed during early gestation, the observed MHC-I expression in the cloned pregnancies is likely to have induced a maternal lymphocytic response that would be detrimental to maintaining viability of the cloned pregnancy. These findings support a role for immunologic rejection in the syndrome of early embryonic loss in cloned bovine pregnancies.

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