Makoto Fujiki

Microminipig, a Non-rodent Experimental Animal Optimized for Life Science Research: Novel Atherosclerosis Model Induced by High Fat and Cholesterol Diet

Atherosclerotic lesions were observed in male and ovariectomized female Microminipig (MMP) fed a high fat and cholesterol diet with sodium cholate (HFCD/SC) for 3 months. HFCD/SC induced hypercholesterolemia accompanied by an increase in serum total cholesterol (T-Cho), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and cholesterol ester (CE). Unlike the mouse or rabbit, a dominant LDL-C fraction in the intact MMP, similar to that in humans, was observed by serum lipoprotein analysis. HFCD/SC increased body weight gain. At the end of the experiment, computed tomography scans of conscious animals showed that HFCD/SC had decreased liver attenuation values (Hounsfield unit) and increased subcutaneous and abdominal fat, suggesting the induction of fatty liver and obesity. HFCD/SC induced atherosclerotic lesions in systemic arteries, including the external and internal iliac arteries, abdominal aorta, coronary artery, and cerebral arterial circle. Atherosclerosis and pathological findings induced by HFCD/SC in MMP were similar to those in humans. The MMP is a potentially suitable tool for investigating human atherosclerosis.

Serum level of cartilage oligomeric matrix protein (COMP) in equine osteoarthritis

This study was designed to assay and compare cartilage oligomeric matrix protein (COMP) in horse sera, in samples from normal and joint diseased horses, and to investigate the relationships between COMP in sera and synovial fluids (SF) with keratan sulphate (KS) data. Sera from 38 horses free of any joint pathology (controls) and from horses with aseptic joint disease (AJD horses, n = 40) were assayed for COMP and KS concentrations. Of the 78 horses in the study, 53 were also assayed for COMP and KS concentrations in SF. COMP and KS were measured by inhibition ELISA, using monoclonal antibodies 12C4 and 5D4, respectively. The COMP concentration in sera from AJD horses (mean +/- s.d. 10.7 +/- 7.4 microg/ml) was significantly (P<0.02) lower than in control sera (14.8 +/- 7.8 microg/ml). The joint disease sera also had significantly lower (P<0.01) KS levels (180.5 +/- 61.8 ng/ml) than controls (237.1 +/- 116.1 ng/ml). A significant correlation (r = 0.52, n = 53, P<0.001) was seen between serum and SF in COMP levels; no such relationship was seen in KS levels. It is possible that serum COMP concentration could be a more specific marker of equine joint disease than any other described to date.

Analysis of cartilage oligomeric matrix protein (COMP) degradation and synthesis in equine joint disease

REASONS FOR PERFORMING STUDY Cartilage oligomeric matrix protein (COMP) is abundant within cartilage; its turnover and/or degradation have been investigated in various equine joint diseases and it has been suggested that COMP fragmentation might be useful for monitoring such conditions. OBJECTIVES To determine whether COMP metabolism is compromised in equine osteoarthritis (OA) and whether COMP degradation is a useful joint marker representing cartilage destruction. HYPOTHESIS A monoclonal antibody (mAb) with a higher affinity for degraded COMP allows discrimination of diseased joints by quantifying COMP levels and fragmentation. METHODS A mAb (clone14G4) was generated against equine cartilage COMP. The NH2-terminal sequence of enzyme-cut COMP fragments recognised by 14G4 was determined, as was the efficiency of binding to COMP (using a generated COMP peptide). COMP concentration and fragmentation were analysed in synovial fluid (SF) from normal horses and those with OA. RESULTS The mAb 14G4 had a higher affinity for the smaller fragments of equine COMP, compared with a mAb (clone 12C4) generated against human COMP. The 14G4 epitope was identified as between C134 and F147. The COMP values in OA (mean +/- s.d. 205.8 +/- 90.9 microg/ml) were significantly higher than in the normal (133.1 +/- 31.5 microg/ml) SF. On the immunoblots of OA sample, the proportions of intact COMP were significantly lower, while smaller fragments ranging from 75 to 290 kDa were higher compared with the normal SF. CONCLUSIONS AND POTENTIAL RELEVANCE The mAb 14G4 reliably detects COMP degradation as well as synthesis, and fragmentation analysis combined with quantification in SF could be useful to study equine OA.

Comparative study of chronic kidney disease in dogs and cats: Induction of myofibroblasts

We investigated the kidneys of dogs and cats to clarify whether renal myofibroblasts induction is associated with the severity of chronic kidney disease (CKD). Immunohistochemical expression of myofibroblast markers, alpha-smooth muscle actin (SMA) and vimentin, were evaluated quantitatively. The degrees of glomerulosclerosis, glomerular hypertrophy, interstitial cell infiltration, and interstitial fibrosis were also evaluated quantitatively. The plasma creatinine (pCre) concentrations correlated with glomerulosclerosis, cell infiltration, and fibrosis in dogs, and only with fibrosis in cats. The alpha-SMA expression correlated with pCre, glomerulosclerosis, cell infiltration, and fibrosis in dogs, and with pCre and fibrosis in cats. Tubular vimentin expression correlated with fibrosis in cats, but not in dogs. Interstitial vimentin expression correlated with pCre, glomerulosclerosis, cell infiltration, and fibrosis in dogs, but only with pCre in cats. In conclusion, it was suggested that the severity of CKD in dogs and cats was mediated by different pathways associated with myofibroblasts expression.

Matrix metalloprotease-9 activity in the cerebrospinal fluid and spinal injury severity in dogs with intervertebral disc herniation.

We investigated whether matrix metalloproteinase (MMP)-9 expression in the cerebrospinal fluid (CSF) of dogs with intervertebral disc herniation (IVDH) is associated with the severity of neurological signs and prognosis. CSF from the cisterna magna (C-CSF) and the lumbar spine (L-CSF) of 34 dogs with IVDH was analyzed using zymography. Activity of MMP-9 in L-CSF was detected in 6 of 34 dogs with IVDH, often for more than 7 days after injury. MMP-9 activity was not detected from any of the C-CSF samples. Of the six cases that were MMP-9 positive, all four cases with grade V that had loss of deep pain were non-ambulatory 6 months after treatment. The remaining two cases with grade III and IV could recover mobility. In dogs with grade V thoracolumbar IVDH, MMP-9 expression in the CSF may indicate severe spinal cord injury with poor prognosis.

A comparative study of chronic kidney disease in dogs and cats: induction of cyclooxygenases.

The present study investigated whether renal cyclooxygenase (COX) induction is associated with the severity of chronic kidney disease (CKD) in dogs and cats. The collected kidneys were examined histopathologically and immunohistochemically. The immunoreactivities of COX-1 and COX-2 were evaluated quantitatively, and the correlations to the plasma creatinine concentrations, glomerular size, glomerulosclerosis, interstitial fibrosis, and interstitial cell infiltration were evaluated statistically. Immunoreactivities for COX-1 were heterogeneously observed in the medullary distal tubules and collecting ducts; no correlations with the severity of renal damage were detected. Immunoreactivities for COX-2 were heterogeneously observed in the macula densa (MD) regions. In dogs, the percentage of COX-2-positive MD was significantly correlated with the glomerular size. In cats, glomeruli with COX-2-positive MD had significantly higher sclerosis scores than those with COX-2-negative MD. In conclusion, renal COX-2 is induced in canine and feline CKD, especially in relation to the glomerular changes.

Cartilage oligomeric matrix protein in canine spinal cord appears in the cerebrospinal fluid associated with intervertebral disc herniation.

Study Design. Cerebrospinal fluid (CSF) from dogs with intervertebral disc herniation (IVDH) were analyzed using a specific antibody against cartilage oligomeric matrix protein (COMP). Immunolocalization and genetic expression of COMP were also examined in the spinal cords of mouse, rat, and dog. Objective. To investigate the hypothesis that COMP is present in CSF of dogs with IVDH, and the clinical relevance of COMP expression in CSF as a potential biomarker of spinal cord injury. Summary of Background Data. In dog IVDH, diagnostic imaging is useful for determining the spinal cord compression, but less useful for assessment of traumatic degeneration. Aggrecan, a well-known component of cartilage matrix, is increased in CSF from cases of neural damage and inflammation of the spinal cord. Methods. CSF from the cisterna magna (C-CSF) and the lumbar spine (L-CSF) of 19 dogs with IVDH and 5 normal dogs were analyzed using inhibition enzyme-linked immunosorbent assay (ELISA) and immunoblotting with an antibody cross-reactive with dog COMP. Samples of normal spinal cord from mouse, rat, and dog were also prepared for immunohistochemistry and in situ hybridization. Results. ELISA values were significantly higher for L-CSF than for C-CSF in dogs with IVDH, whereas there was no significant difference between them in normal dogs. Immunoblots of L-CSF samples revealed positive bands of approximately 500 kDa in 6 cases of IVDH (positive cases), but no signal in negative cases. ELISA values were significantly higher in the positive cases than in the negative cases. Both COMP protein and mRNA were present at high levels in the gray matter of the spinal cord in all species. Conclusion. In dog IVDH, release of COMP from the spinal cord in association with injury may lead to COMP accumulation in L-CSF posterior to the site of disc extrusion, and therefore might be a predictive marker of spinal cord injury.

Immunohistochemical analysis of cyclooxygenase-2 induction during wound healing in dog skin

Cyclooxygenase (COX)-2 is an inducible isoform of COX and is expressed under abnormal health conditions. This study elucidated the cutaneous induction of COX-2 during the wound healing processes in dog skin. Dog skin was sutured after punch biopsy and investigated histologically and immunohistochemically on days 0 (normal), 1, 3, 5, 7, 10, and 14 after injury. Histological changes, including infiltration of inflammatory cells and proliferation of fibroblast-like cells, were observed as predicted, and there was a close and significant correlation between these 2 events. COX-2-positive cells were detected in the epidermis between days 1 and 7, and bimodal peaks were observed in the case of the percentage of COX-2-positive cells. In inflammatory cells, COX-positive signals were detected on day 3 only. Here, we clarified the localization and pattern of the induced COX-2 expression during wound healing in dog skin.

Characteristics and multipotency of equine dedifferentiated fat cells

ABSTRACT Dedifferentiated fat (DFAT) cells have been shown to be multipotent, similar to mesenchymal stem cells (MSCs). In this study, we aimed to establish and characterize equine DFAT cells. Equine adipocytes were ceiling cultured, and then dedifferentiated into DFAT cells by the seventh day of culture. The number of DFAT cells was increased to over 10 million by the fourth passage. Flow cytometry of DFAT cells showed that the cells were strongly positive for CD44, CD90, and major histocompatibility complex (MHC) class I; moderately positive for CD11a/18, CD105, and MHC class II; and negative for CD34 and CD45. Moreover, DFAT cells were positive for the expression of sex determining region Y-box 2 as a marker of multipotency. Finally, we found that DFAT cells could differentiate into osteogenic, chondrogenic, and adipogenic lineages under specific nutrient conditions. Thus, DFAT cells could have clinical applications in tissue regeneration, similar to MSCs derived from adipose tissue.

Osteochondral Regeneration of the Loading-bearing Site Using a Scaffold Free Three-dimensional Construct of Adipose Tissue-derived Mesenchymal Stem Cells in Pigs

Background: Many surgical strategies for reconstruction of both bone and cartilage have ever been investigated to restore joint structure and function in the late stages of Osteoarthritis (OA). This study was designed to investigate the regeneration of articular cartilage and subchondral bone in the loading-bearing site using a three-dimensional (3D) construct of autologous adipose tissue-derived mesenchymal stem cells (AT-MSCs). Methods: A 3D construct consisting of approximately 1,920 spheroids each containing 5.0 × 104 AT-MSCs was implanted into an osteochondral defect (with a diameter of 6.8 mm and a depth of 6 mm) in the right femoral medial condyle in five adult mini-pigs. The contralateral (left femoral) defect was the control. At three and six months post-operatively, the defects were evaluated using both CT and MR imaging. The radiolucent volume (RV, mm3) of the defects was calculated based on the multiplanar reconstruction of the CT images. MR images and gross and histologic pathology features were scored using a modified-MOCART system and the ICRS system, respectively, at six months post-operatively. Results: The percentages of RVs at three and six months compared with those immediately after the surgeries were significantly decreased in the implanted defects compared with the control defects. The total scores of modified- MOCART system were also significantly increased in the implanted sites comparing to the controls. Although there were no statistical differences in the average of gross scores, the average histological scores were significantly higher in the implanted sites than in the control sites. Conclusion: This is the first report suggesting that implantation of a scaffold-free three dimensional construct of only AT-MSCs into the osteochondral defect regenerates the original cartilage and subchondral bone structures over six months post-operatively in the loading-bearing site of large animal.