Animal Models of Human Disease 2.0

Rat animal models are widely used owing to their relatively superior cognitive abilities and higher similarity compared with mouse models to human physiological characteristics. However, their use is limited because of difficulties in establishing embryonic stem cells and performing genetic modifications, and insufficient embryological research. In this study, we established optimal superovulation and fertilized–egg transfer conditions, including optimal hormone injection concentration (≥150 IU/kg of PMSG and hCG) and culture medium (mR1ECM), to obtain high-quality zygotes and establish in vitro fertilization conditions for rats. Next, sgRNA with optimal targeting activity was selected by performing PCR analysis and the T7E1 assay, and the CRISPR/Cas9 system was used to construct a rat model for muscular dystrophy by inducing a deficiency in the fukutin gene without any off-target effect detected. The production of fukutin knockout rats was phenotypically confirmed by observing a drop-in body weight to one-third of that of the control group. In summary, we succeeded in constructing the first muscular dystrophy disease rat model using the CRISPR/CAS9 system for increasing future prospects of producing various animal disease models and encouraging disease research using rats. Full article

A deficiency in the shortest dystrophin-gene product, Dp71, is a pivotal aggravating factor for intellectual disabilities in Duchenne muscular dystrophy (DMD). Recent advances in preclinical research have achieved some success in compensating both muscle and brain dysfunctions associated with DMD, notably using exon skipping strategies. However, this has not been studied for distal mutations in the DMD gene leading to Dp71 loss. In this study, we aimed to restore brain Dp71 expression in the Dp71-null transgenic mouse using an adeno-associated virus (AAV) administrated either by intracardiac injections at P4 (ICP4) or by bilateral intracerebroventricular (ICV) injections in adults. ICP4 delivery of the AAV9-Dp71 vector enabled the expression of 2 to 14% of brain Dp71, while ICV delivery enabled the overexpression of Dp71 in the hippocampus and cortex of adult mice, with anecdotal expression in the cerebellum. The restoration of Dp71 was mostly located in the glial endfeet that surround capillaries, and it was associated with partial localization of Dp71-associated proteins, α1-syntrophin and AQP4 water channels, suggesting proper restoration of a scaffold of proteins involved in blood–brain barrier function and water homeostasis. However, this did not result in significant improvements in behavioral disturbances displayed by Dp71-null mice. The potential and limitations of this AAV-mediated strategy are discussed. This proof-of-concept study identifies key molecular markers to estimate the efficiencies of Dp71 rescue strategies and opens new avenues for enhancing gene therapy targeting cognitive disorders associated with a subgroup of severely affected DMD patients. Full article

The Adriamycin (ADR) nephropathy model, which induces podocyte injury, is limited to certain mouse strains due to genetic susceptibilities, such as the Prkdc^R2140C polymorphism. The FVB/N strain without the R2140C mutation resists ADR nephropathy. Meanwhile, a detailed analysis of the progression of ADR nephropathy in the FVB/N strain has yet to be conducted. Our research aimed to create a novel mouse model, the FVB-Prkdc^R2140C, by introducing Prkdc^R2140C into the FVB/NJcl (FVB) strain. Our study showed that FVB-Prkdc^R2140C mice developed severe renal damage when exposed to ADR, as evidenced by significant albuminuria and tubular injury, exceeding the levels observed in C57BL/6J (B6)-Prkdc^R2140C. This indicates that the FVB/N genetic background, in combination with the R2140C mutation, strongly predisposes mice to ADR nephropathy, highlighting the influence of genetic background on disease susceptibility. Using RNA sequencing and subsequent analysis, we identified several genes whose expression is altered in response to ADR nephropathy. In particular, Mmp7, Mmp10, and Mmp12 were highlighted for their differential expression between strains and their potential role in influencing the severity of kidney damage. Further genetic analysis should lead to identifying ADR nephropathy modifier gene(s), aiding in early diagnosis and providing novel approaches to kidney disease treatment and prevention. Full article

High-grade hemorrhoids are usually recommended to receive operational treatments. However, these traditional surgeries are associated with severe postoperative pain. A procedure for prolapse and hemorrhoids (PPH), a circular staple device, has been developed to improve short-term outcomes, including reducing the severity of postoperative pain. PPH, compared to conventional surgery, has been associated with the incidence of anatomical anal stenosis. The causes of stenosis after PPH are not yet clear. We first analyzed the complications of our patients with PPH, and then developed a rat model to verify the tension force of PPH using Hematoxylin-eosin, Masson’s trichrome, immunohistochemistry, and immunofluorescence staining. Our clinical data showed that PPH significantly improved postoperative pain, but that it resulted in higher incidences of complications, including anal stenosis, than hemorrhoidectomy. We simulated the status of PPH and developed a rat model to verify PPH’s tension force, including the scarring area and the deposition of proinflammatory factors, angiogenic factors, and fibrotic factors. The tension wound histological data showed more extensive granulation tissue and inflammatory cell infiltration and a thicker epidermis than the control group on day 12 post-operation and tension treatment. In addition to IL-1β and IL-10 cytokines on day 3 and IL-1β, IL-6, and IL-10 cytokines on day 12 post-operation in the tension group, two angiogenic factors, CD31 and VEGF-A, were found to have a more significant expression on day 7 post-operation in the tension group. The mean scar area was larger and the distribution of fibrotic proteins (collagen 1, α-SMA, CTGF, and MMP2) in the tension group was significantly broader than in the control on day 12 post-operation and tension treatment. Based on the findings of our animal model, the development of a lesser tensile force for PPH to decrease the deposition of proinflammatory factors, angiogenic factors, and fibrotic factors is urgently required. Full article

Background: Coronaviral infection-induced acute lung injury has become a major threat to public health, especially through the ongoing pandemic of COVID-19. Apta-1 is a newly discovered Aptamer that has anti-inflammatory effects on systemic septic responses. The therapeutic effects of Apta-1 on coronaviral infection-induced acute lung injury and systemic responses were evaluated in the present study. Methods: Female A/J mice (at 12–14 weeks of age) were challenged with murine hepatitis virus 1 (MHV-1), a coronavirus, at 5000 PFU intranasally, followed by Apta-1 intravenously administered (100 mg/kg, twice) 1.5 h or 2 days after viral delivery. Animals were sacrificed at Day 2 or Day 4. Lung tissues were examined with H&E, immunohistochemistry staining, and western blotting. RT-qPCR was used for cytokine gene expression. Serum and plasma were collected for laboratory assessments. Results: Apta-1 treatment reduced viral titers, prevented MHV-1-induced reduction of circulating blood volume and hemolysis, reduced alveolar space hemorrhage, and protease-activated receptor 1 (PAR-1) cleavage. Apta-1 treatment also significantly reduced chemokine (MKC, MCP-1, and RANTES) levels, as well as AST, ALT, total bilirubin, and reduced unconjugated bilirubin levels in the serum. Conclusion: Apta-1 showed therapeutic benefits in coronaviral infection-induced hemorrhage and PAR-1 cleavage in the lung. It also has anti-inflammatory effects systemically. Full article

Heterozygous carriers of the glucocerebrosidase 1 (GBA) L444P Gaucher mutation have an increased risk of developing Parkinson’s disease (PD). The GBA mutations result in elevated alpha synuclein (aSyn) levels. Heterozygous mice carrying one allele with the L444P mutation knocked-into the mouse gene show increased aSyn levels and are more sensitive to motor deficits following exposure to the neurotoxin (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) MPTP than wild-type mice. Paraquat (PQ), a herbicide, increases PD risk in most studies. Its effects on the brain involve alterations in the gut microbiome. Exposure to dextran sulfate sodium (DSS), a mouse model of colitis, can be used to determine whether gut microbiome alterations are sufficient to induce PD-relevant phenotypes. We rederived the A53T-L444P and A53T mouse lines to assess whether PQ, PQ in combination with radiation exposure (IR), and DSS have differential effects in A53T and A53T-L444P mice and whether these effects are associated with alterations in the gut microbiome. PQ and PQ + IR have differential effects in A53T and A53T-L444P mice. In contrast, effects of DSS are only seen in A53T-L444P mice. Exposure and genotype modulate the relationship between the gut microbiome and behavioral performance. The gut microbiome may be an important mediator of how environmental exposures or genetic mutations yield behavioral and cognitive impacts. Full article

While the manifestations of many inherited retinal disorders are limited to loss of vision, others are part of a syndrome that affects multiple tissues, particularly the nervous system. Most syndromic retinal disorders are thought to be recessively inherited. Two dogs out of a litter of Cirneco dell′ Etna dogs, both males, showed signs of retinal degeneration, along with tremors and signs described as either atypical seizures or paroxysmal dyskinesias, while the other two male littermates were normal. We named this oculo-neurological syndrome CONS (Cirneco oculo-neurological syndrome), and undertook homozygosity mapping and whole-genome sequencing to determine its potential genetic etiology. Notably, we detected a 1-bp deletion in chromosome 6 that was predicted to cause a frameshift and premature stop codon within the canine AMPD2 gene, which encodes adenosine monophosphate deaminase, an enzyme that converts adenosine 5′-monophosphate (AMP) to inosine 5’-monophosphate (IMP). Genotyping of the available Cirneco population suggested perfect segregation between cases and controls for the variant. Moreover, this variant was absent in canine genomic databases comprised of thousands of unaffected dogs. The AMPD2 genetic variant we identified in dogs presents with retinal manifestations, adding to the spectrum of neurological manifestations associated with AMPD2 variants in humans. Full article

Gulf War (GW) veterans show gastrointestinal disturbances and gut dysbiosis. Prolonged antibiotic treatments commonly employed in veterans, especially the use of fluoroquinolones and aminoglycosides, have also been associated with dysbiosis. This study investigates the effect of prolonged antibiotic exposure on risks of adverse renal pathology and its association with gut bacterial species abundance in underlying GWI and aims to uncover the molecular mechanisms leading to possible renal dysfunction with aging. Using a GWI mouse model, administration of a prolonged antibiotic regimen involving neomycin and enrofloxacin treatment for 5 months showed an exacerbated renal inflammation with increased NF-κB activation and pro-inflammatory cytokines levels. Involvement of the high mobility group 1 (HMGB1)-mediated receptor for advanced glycation end products (RAGE) activation triggered an inflammatory phenotype and increased transforming growth factor-β (TGF-β) production. Mechanistically, TGF-β- induced microRNA-21 upregulation in the renal tissue leads to decreased phosphatase and tensin homolog (PTEN) expression. The above event led to the activation of protein kinase-B (AKT) signaling, resulting in increased fibronectin production and fibrosis-like pathology. Importantly, the increased miR-21 was associated with low levels of Lachnospiraceae in the host gut which is also a key to heightened HMGB1-mediated inflammation. Overall, though correlative, the study highlights the complex interplay between GWI, host gut dysbiosis, prolonged antibiotics usage, and renal pathology via miR-21/PTEN/AKT signaling. Full article

Background: The regeneration of severe traumatic muscle injuries is an unsolved medical need that is relevant for civilian and military medicine. In this work, we produced a critically sized nonhealing muscle defect in a mouse model to investigate muscle degeneration/healing phases. Materials and methods: We caused a freeze injury (FI) in the biceps femoris of C57BL/6N mice. From day 1 to day 25 post-injury, we conducted histological/morphometric examinations, an analysis of the expression of genes involved in inflammation/regeneration, and an in vivo functional evaluation. Results: We found that FI activates cytosolic DNA sensing and inflammatory responses. Persistent macrophage infiltration, the prolonged expression of eMHC, the presence of centrally nucleated myofibers, and the presence of PAX7+ satellite cells at late time points and with chronic physical impairment indicated inadequate repair. By looking at stem-cell-based therapeutic protocols of muscle repair, we investigated the crosstalk between M1-biased macrophages and human amniotic mesenchymal stem cells (hAMSCs) in vitro. We demonstrated their reciprocal paracrine effects where hAMSCs induced a shift of M1 macrophages into an anti-inflammatory phenotype, and M1 macrophages promoted an increase in the expression of hAMSC immunomodulatory factors. Conclusions: Our findings support the rationale for the future use of our injury model to exploit the full potential of in vivo hAMSC transplantation following severe traumatic injuries. Full article