Equestrian-Related Musculoskeletal Injuries Presenting to a Chiropractic Practice: A Retrospective Chart Review of 19 Patients.

Objective: The purpose of this study was to describe the types of equestrian-related musculoskeletal injuries and their management. Methods: We retrospectively reviewed the charts of 19 patients who presented with injuries from equestrian activities at a chiropractic practice from December 2000 to December 2020. Deidentified data were extracted from the charts and summarized. Results: Of the 19 patients, 42.3% presented with acute trauma, 38.5% had overuse injuries, and 19.2% had chronic injuries as a result of previous trauma. We found that 90% of overuse injuries and 18.2% of acute injuries led to chronic conditions that needed ongoing management. Conclusion: From this sample of patients, there was a high percentage of overuse and chronic injuries for patients who participated in equestrian activities.

Acrylamide Production in Autoclaved Rodent Feed.

Sterilization of rodent feed by steam autoclaving is a common practice in many research institutions. Often we only considerthe beneficial effects of this process-the reduction of microbial contamination-and forget that the high temperatures andpressures can have negative effects on diet quality. The purpose of our study was to assess both the physical and chemicalchanges to a standard rodent feed autoclaved at multiple sterilization temperatures and the effects of the treated diets on mice. Pelleted NIH31 rodent feed was autoclaved at 4 sterilization temperatures (230, 250, 260, and 270 °F). Feed pellet hardness and the acrylamide concentrations of the diets were tested and compared with irradiated NIH31 feed. Study diets were fed to mice for 28 d, after which tissue samples were collected for analysis of acrylamide, glycidamide (the active metabolite of acrylamide), and genotoxicity. Both feed pellet hardness and acrylamide concentration increased with increasing sterilization temperatures; however, neither affected feed intake or body weight gain. Plasma acrylamide and glycidamide weresignificantly elevated only in mice fed NIH31 diet autoclaved at 270 °F compared with the irradiated feed, whereas urineacrylamide and glycidamide metabolites were significantly elevated in most autoclaved diets. Liver DNA adducts, whichcorrelate with genotoxicity, were significantly elevated in all autoclaved diets compared with the irradiated diet. Institutionsthat autoclave their animal diets should carefully consider the temperatures necessary to achieve feed sterilization and thetype of studies in which these autoclaved diets are used.

Conditional ablation of the RFX4 isoform 1 transcription factor: Allele dosage effects on brain phenotype.

Regulatory factor X4 (RFX4) isoform 1 is a recently discovered isoform of the winged helix transcription factor RFX4, which can bind to X-box consensus sequences that are enriched in the promoters of cilia-related genes. Early insertional mutagenesis studies in mice first identified this isoform, and demonstrated that it was crucial for mouse brain development. RFX4 isoform 1 is the only RFX4 isoform significantly expressed in the mouse fetal and adult brain. In this study, we evaluated conditional knock-out (KO) mice in which one or two floxed alleles of Rfx4 were deleted early in development through the use of a Sox2-Cre transgene. Heterozygous deletion of Rfx4 resulted in severe, non-communicating congenital hydrocephalus associated with hypoplasia of the subcommissural organ. Homozygous deletion of Rfx4 resulted in formation of a single ventricle in the forebrain, and severe dorsoventral patterning defects in the telencephalon and midbrain at embryonic day 12.5, a collection of phenotypes that resembled human holoprosencephaly. No anatomical abnormalities were noted outside the brain in either case. At the molecular level, transcripts encoded by the cilia-related gene Foxj1 were significantly decreased, and Foxj1 was identified as a direct gene target of RFX4 isoform 1. The phenotypes were similar to those observed in the previous Rfx4 insertional mutagenesis studies. Thus, we provide a novel conditional KO animal model in which to investigate the downstream genes directly and/or indirectly regulated by RFX4 isoform 1. This model could provide new insights into the pathogenesis of obstructive hydrocephalus and holoprosencephaly in humans, both relatively common and disabling birth defects.

Targeted disruption of Zfp36l2, encoding a CCCH tandem zinc finger RNA-binding protein, results in defective hematopoiesis.

Members of the tristetraprolin family of tandem CCCH finger proteins can bind to AU-rich elements in the 3'-untranslated region of mRNAs, leading to their deadenylation and subsequent degradation. Partial deficiency of 1 of the 4 mouse tristetraprolin family members, Zfp36l2, resulted in complete female infertility because of early embryo death. We have now generated mice completely deficient in the ZFP36L2 protein. Homozygous Zfp36l2 knockout (KO) mice died within approximately 2 weeks of birth, apparently from intestinal or other hemorrhage. Analysis of peripheral blood from KO mice showed a decrease in red and white cells, hemoglobin, hematocrit, and platelets. Yolk sacs from embryonic day 11.5 (E11.5) Zfp36l2 KO mice and fetal livers from E14.5 KO mice gave rise to markedly reduced numbers of definitive multilineage and lineage-committed hematopoietic progenitors. Competitive reconstitution experiments demonstrated that Zfp36l2 KO fetal liver hematopoietic stem cells were unable to adequately reconstitute the hematopoietic system of lethally irradiated recipients. These data establish Zfp36l2 as a critical modulator of definitive hematopoiesis and suggest a novel regulatory pathway involving control of mRNA stability in the life cycle of hematopoietic stem and progenitor cells.

Abnormal glucose metabolism in heterozygous mutant mice for a type I receptor required for BMP signaling.

BMPRIA and its high-affinity ligand BMP4 have recently been shown to be expressed in the beta-cells of the pancreas. Here, we report the abnormalities of heterozygous mice for Bmpr1a in glucose metabolism during the course of intraperitoneal glucose tolerance test. The heterozygous mice had increased blood glucose levels throughout the first 2.5 h after the administration of glucose. Analysis of glucose-stimulated insulin secretion (GSIS) indicates that insulin secretion in the heterozygous mice is compromised, and induction of secreted insulin by stimulation is substantially lower compared with the wild-type controls. No apparent abnormalities in pancreas, thyroid, and liver were seen upon histological examination. Real-time PCR results of selected genes showed an increase in the mRNA level of Ins1 and Ins2 in the heterozygous group. These results indicate that the glucose-sensing pathway in these heterozygous mice is altered because of the heterozygosity in Bmpr1a. Together, our data suggest that BMP signaling through BMPRIA plays an important role in glucose metabolism and possibly working through the GSIS pathway.

Generation of a conditional null allele for Dmp1 in mouse.

Dentin matrix protein1 (DMP1), highly conserved in humans and mice, is highly expressed in teeth, the skeleton, and to a lesser extent in nonskeletal tissues such as brain, kidney, and salivary gland. Pathologically, DMP1 is associated with several forms of cancers and with tumor-induced osteomalacia. Conventional disruption of the murine Dmp1 gene results in defects in dentin in teeth and in the skeleton, including hypophosphatemic rickets, and abnormalities in phosphate homeostasis. Human DMP1 mutations are responsible for the condition known as autosomal recessive hypophosphatemic rickets. For better understanding of the roles of DMP1 in different tissues at different stages of development and in pathological conditions, we generated Dmp1 floxed mice in which loxP sites flank exon 6 that encodes for over 80% of DMP1 protein. We demonstrate that Cre-mediated recombination using Sox2-Cre, a Cre line expressed in epiblast during early embryogenesis, results in early deletion of the gene and protein. These homozygous Cre-recombined null mice display an identical phenotype to conventional null mice. This animal model will be useful to reveal distinct roles of DMP1 in different tissues at different ages.

Spontaneous and irradiation-induced tumor susceptibility in BRCA2 germline mutant mice and cooperative effects with a p53 germline mutation.

Mutations in both p53 and BRCA2 are commonly seen together in human tumors suggesting that the loss of both genes enhances tumor development. To elucidate this interaction in an animal model, mice lacking the carboxy terminal domain of Brca2 were crossed with p53 heterozygous mice. Females from this intercross were then irradiated with an acute dose of 5 Gy ionizing radiation at 5 weeks of age and compared to nonirradiated controls. We found decreased survival and timing of tumor onsets, and significantly higher overall tumor incidences and prevalence of particular tumors, including stomach tumors and squamous cell carcinomas, associated with the homozygous loss of Brca2, independent of p53 status. The addition of a p53 mutation had a further impact on overall survival, incidence of osteosarcomas and stomach tumors, and tumor latency. The spectrum of tumors observed for this Brca2 germline mouse model suggest that it faithfully recapitulates some human disease phenotypes associated with BRCA2 loss. In addition, these findings include extensive in vivo data demonstrating that germline Brca2 and p53 mutations cooperatively affect animal survivals, tumor susceptibilities, and tumor onsets.

Mutant Brca2/p53 mice exhibit altered radiation responses in the developing mammary gland.

Appropriate balance between proliferation and apoptosis is critical for mammary gland development and is often altered during tumorigenesis. Carcinogens like radiation induce DNA damage and activate protective responses such as cell cycle arrest and apoptosis. We used mice carrying Brca2(-/-) and/or p53(-/-) mutations to evaluate the individual and combined effects of these genes on cell proliferation and apoptosis in the developing mammary gland. Mice were exposed to 5Gy of radiation or chamber exposure (controls) followed by injection with BrdU. Mammary glands were collected 6 h post-radiation exposure and evaluated for proliferation (BrdU) and apoptosis (TUNEL) in terminal end buds (TEB) and ducts. Under control conditions, the Brca2 mutation reduced proliferation and apoptosis in TEB but not ducts, whereas the p53 mutation reduced apoptosis in TEB and ducts but did not influence proliferation. Despite these alterations in proliferation and/or apoptosis, neither mutation, either individually or combined, significantly altered the overall balance between the two as measured by the proliferation to apoptosis ratio (growth index). Following irradiation, the Brca2 mutation had no significant effect on proliferation or apoptosis, whereas the p53 mutation resulted in reduced apoptosis in TEB and ducts but did not significantly influence proliferation. Neither mutation by itself altered the growth index in the TEB after irradiation although combined Brca2/p53 mutation caused significantly increased proliferation, reduced apoptosis, and an elevated growth index in TEB and ducts. These results reveal both independent and collaborative growth regulatory roles for Brca2 and p53 under normal and adverse environmental conditions. Additionally, we demonstrate the importance of gene-environment interactions by showing that Brca2- and p53-deficient mice can compensate for their genetic deficiencies under control conditions but not after exposure to radiation. We also demonstrate distinct spatial differences in the cellular functions of Brca2 and p53 and show that combined mutation of both genes is more detrimental than loss of either gene alone.

Cancer susceptibility of mice with a homozygous deletion in the COOH-terminal domain of the Brca2 gene.

Inherited mutations of the human BRCA2 gene confer increased risks for developing breast, ovarian, and several other cancers. Unlike previously described Brca2 knockout mice that display predominantly embryonic lethal phenotypes, we developed mice with a homozygous germ-line deletion of Brca2 exon 27 that exhibit a moderate decrease in perinatal viability and are fertile. We deleted this Brca2 COOH-terminal domain because it interacts directly with the Rad51 protein, contains a nuclear localization signal, and is required to maintain genomic stability in response to various types of DNA damage. These homozygous Brca2-mutant mice have a significantly increased overall tumor incidence and decreased survival compared with their heterozygous littermates. Virgin female mice homozygous for this Brca2 mutation also display an inhibition of ductal side branching in the mammary gland at 6 months of age. Given their substantial viability and cancer predisposition, these mutant mice will be useful to further define the role of the COOH-terminal Brca2 domain in tumorigenesis both in vivo and in vitro.

Arachidonic and linoleic acid metabolism in mouse intestinal tissue: evidence for novel lipoxygenase activity.

Previous studies in our laboratory revealed a high expression of 15-lipoxygenase-1 in human colorectal carcinomas, suggesting the importance of lipoxygenase in colorectal tumor development. In this report, we have investigated the metabolism of arachidonic and linoleic acid by intestinal tissues of Min mice, an animal model for intestinal neoplasia. The polyp and normal tissues from Min mice intestine were homogenized, incubated with arachidonic or linoleic acid, and analyzed by reverse-, straight-, and chiral-phase HPLC. Arachidonic acid was converted to prostaglandins E2 and F2alpha. Little 12- or 15-hydroxyeicosatetraenoic acid was detected. Cyclooxygenase (COX)-2 was detected in polyps and the adjacent normal tissues by Western immunoblotting, but neither COX-1 nor leukocyte-type 12-lipoxygenase, the murine ortholog to human 15-lipoxygenase-1, was detected. These tissue homogenates converted linoleic acid to an equal mixture of 9(S)- and 13(S)-hydroxyoctadecadienoic acid (HODE). Inhibition of lipoxygenase activity with nordihydroguaiaretic acid blocked HODEs formation, but the COX inhibitor indomethacin did not. Degenerative-nested PCR analyses using primers encoded by highly conserved sequences in lipoxygenases detected 5-lipoxygenase, leukocyte-type 12-lipoxygenase, platelet-type 12-lipoxygenase, 8-lipoxygenase, and epidermis-type lipoxygenase-3 in mouse intestinal tissue. All of these PCR products represent known lipoxygenase that are not reported to utilize linoleic acid preferentially as substrate and do not metabolize linoleic acid to an equal mixture of 9(S)- and 13(S)-HODE. This somewhat unique profile of linoleate product formation in Min mice intestinal tissue suggests the presence of an uncharacterized and potentially novel lipoxygenase(s) that may play a role in intestinal epithelial cell differentiation and tumor development.