Gene expression profile in bone of diabetes-prone BB/OK rats fed a high-fat diet.

A high-fat diet (HFD) has been recognized as a risk factor for diseases such as dyslipidemia, atherosclerosis, obesity, and osteoporosis. However, studies analyzing gene expression after HFD in bone are rare. That prompted us to analyze the expression of selected genes in bone of 4-week-old diabetes-prone B(io)B(reeding) rats. Two breeding pairs were fed a HFD (+10 % tallow) or were fed a normal diet (ND; Ssniff R-Z) before mating and afterward during pregnancy. After the birth of progeny, parents continued to be given HFD or ND until the progeny was weaned (3 weeks). Thereafter, offspring were weaned and were fed the same food as their parents up to an age of 4 weeks. Body weight was measured at an age of 4 weeks, and subsequently 13 HFD rats and 13 ND rats were killed and the tibial bone was harvested to analyze the expression of 53 genes in bone. All rats fed HFD were significantly heavier than rats fed ND after 3 and 4 weeks. The diet also influenced the expression of genes in bone. There were significant differences in 20 out of 53 genes studied between rats fed HFD compared with rats fed ND. Four out of 20 had a lower and 17 out of 20 genes a higher expression in HFD rats, but differences in gene expression showed obvious differences between males and females. There were only two genes that were similarly different between males and females: Bmp4 and Atf4. Two genes, Foxg1 and Npy, were inversely expressed in males and females. It seems that the gene expression is differently regulated by diet during pregnancy and later in life between males and females. Nevertheless, it cannot be excluded that HFD also acts as an epigenetic factor in the development of offspring in utero.

Histomorphometric and radiographical changes after lumbar implantation of the PEEK nonfusion interspinous device in the BB.4S rat model.

STUDY DESIGN: An experimental animal study. OBJECTIVE: To investigate histomorphometric and radiographical changes in the BB.4S rat model after PEEK (polyetheretherketone) nonfusion interspinous device implantation. SUMMARY OF BACKGROUND DATA: Clinical effectiveness of the PEEK nonfusion spine implant Wallis (Abbott, Bordeaux, France; now Zimmer, Warsaw, IN) is well documented. However, there is a lack of evidence on the long-term effects of this implant on bone, in particular its influence on structural changes of bone elements of the lumbar spine. METHODS: Twenty-four male BB.4S rats aged 11 weeks underwent surgery for implantation of a PEEK nonfusion interspinous device or for a sham procedure in 3 groups of 8 animals each: (1) implantation at level L4-L5; (2) implantation at level L5-L6; and (3) sham surgery. Eleven weeks postoperatively osteolyses at the implant-bone interface were measured via radiograph, bone mineral density of vertebral bodies was analyzed using osteodensitometry, and bone mineral content as well as resorption of the spinous processes were examined by histomorphometry. RESULTS.: Resorption of the spinous processes at the site of the interspinous implant was found in all treated segments. There was no significant difference in either bone density of vertebral bodies or histomorphometric structure of the spinous processes between adjacent vertebral bodies, between treated and untreated segments and between groups. CONCLUSION: These findings indicate that resorption of spinous processes because of a result of implant loosening, inhibit the targeted load redistribution through the PEEK nonfusion interspinous device in the lumbar spinal segment of the rat. This leads to reduced long-term stability of the implant in the animal model. These results suggest that PEEK nonfusion interspinous devices like the Wallis implants may have time-limited effects and should only be used for specified indications.

Phenotypic and gene expression differences between DA, BN and WOKW rats.

BACKGROUND: Because inbred rat strains are widely used as laboratory models, knowledge of phenotypic and genetic variations between strains will be useful to obtain insight into the relationship between different strains. METHODS AND RESULTS: We studied phenotypic traits: of each strain--BN/K, DA/K and WOKW--10 male rats were studied for body weight and serum constituents at an age of 10 and 30 weeks. In addition, a total of 95 rats were studied for life expectancy. At an age of 30 weeks, these male rats were killed by an overdose of anesthetic (Sevofluran, Abbott), and the subcutaneous and visceral adipose tissue as well as bone tissue were removed to study the expression of 20 genes. There were significant differences in body weight, serum lipids and leptin at an age of 30 weeks between strains. Regarding life expectancy, BN rats lived longest (1072±228d). The highest gene expression was found in bone of BN rats. In adipose tissues, Nfkb1 is only expressed in subcutaneous adipocytes, and 5 genes, Col2a1, Mmp9, Tnfa, Ins1 and Cyp24a1, are not expressed in adipocytes. The ranking BN = DA>WOKW was observed in only one gene in subcutaneous (Fto) and visceral adipocytes (Col6a1). There were no significant differences in gene expression of one gene in subcutaneous adipocytes and of 3 genes in visceral adipocytes. Comparing the gene expression in visceral and subcutaneous adipocytes, only one gene showed a comparable behavior (Bmp1). CONCLUSION: From these results, it can be concluded that obvious phenotypic differences are caused by genetic differences between three rat strains, BN, DA and WOKW, as supported by gene expression studies in bone and adipose tissues. Especially BN rats can be used to study the genetic basis of long life.

Gene expression profiling supports the role of Repin1 in the pathophysiology of metabolic syndrome.

Congenic BB rat strains carrying a SHR segment (D4Got41-Tacr1; 60.5-122.8 Mb; BB.4S) or a WOKW segment (D4Got41-Fabp1; 60.5-104.6 Mb; BB.4W) of chromosome 4 within the BB/OK background develop facets of the metabolic syndrome when compared with their parental BB/OK rats. To narrow down potential genes involved in the pathophysiology of metabolic syndrome, gene expression studies in adipose tissues of BB/OK, BB.4S, and BB.4W rats were initiated. Total RNA of subcutaneous and epididymal adipose tissue of BB/OK (n=10), congenic BB.4S (n=8), and BB.4W (n=9) males at an age of 4 weeks was isolated. The mRNA expression of 92 genes involved in obesity, insulin resistance and other metabolic traits was measured by RT-PCR. Significant differences in gene expression were only found in Repin1 in both adipose tissues. Congenic BB.4W showed significantly lower gene expression than did BB.4S and BB/OK. Our findings and newly published findings of Repin1 in 3T3-L1 adipocytes support the hypothesis that Repin1 may affect the development of facets of the metabolic syndrome.

High-fat diet protects BB/OK rats from developing type 1 diabetes.

BACKGROUND: It is well known that lipid metabolism plays an important role in the early stages of type 1 diabetes (T1D). For that reason, we examined factors that influence lipid metabolism of BioBreeding/Ottawa Kalsburg (BB/OK) rats that spontaneously develop an insulin-dependent T1D. METHODS: BB/OK female rats were fed a high-fat diet during pregnancy (Ssniff R-Z + 10% tallow) and their progeny were also given this diet up to an age of 30 weeks (n = 55) or 4 weeks (n = 14) to study gene expression of Pparg, Fasn, Lep, Adipoq, Repin1, Rarres 2, and Glut4 in adipose tissue. Forty-two BB/OK rats fed the normal diet (Ssniff R-Z) during pregnancy and the observation period served as controls. RESULTS: The high-fat diet significantly decreased diabetes frequency in BB/OK rats when compared with control rats (71 versus 95%, p = 0.002). Although this difference was also reflected in the male rats (68 versus 100%, p = 0.003), no significant variation was observed in female rats (73 versus 90%, p = 0.23). The high-fat diet resulted in significantly reduced mRNA expression of examined genes in subcutaneous adipose tissue, but not in visceral adipose tissue, except for Fasn and Repin1 expression. CONCLUSIONS: A high-fat diet seems to protect BB/OK rats from T1D in a sex-specific manner. The data suggest that a high-fat diet might influence fat accumulation and/or fat metabolism and prevent T1D development in male rats, which is supported by changes in adipose tissue gene expression.

Transplantation of adipose tissue protects BB/OK rats from type 1 diabetes development.

B(io) B(reedding)/O(ttawa) K(alsburg) rats spontaneously develop insulin-dependent type 1 diabetes. Days before BB/OK rats become diabetic, their body seems to be flabby which may be attributed to loss of subcutaneous fat. However, the rats are normoglycemic and manifest 3-4 days later. This observation prompted us to search for possibilities to avoid the loss of adipose tissue. BB/OK rats were subcutaneously grafted with visceral adipose tissue. In total, 34 (71%) out of 48 male and 23 (49%) out of 47 female BB/OK rats grafted with adipose tissue developed type 1 diabetes so that significantly more females than males were protected from diabetes development (p=0.03). In the control group, 17 (85%) out of 20 male and 20 (95%) out of 21 female BB/OK rats were diabetic. Adipose tissue transplantation can protect BB/OK rats from type 1 diabetes development in a sex specific manner. One could conclude that the manipulations have influenced fat accumulation and/or fat metabolism which prevent type 1 diabetes development in about 50% of BB/OK rats. This idea is supported by the finding that a mutation in the leptin receptor of NOD mice suppresses type 1 diabetes progression.

Is there an autoimmune process in bone? Gene expression studies in diabetic and nondiabetic BB rats as well as BB rat-related and -unrelated rat strains.

It is well known that type 1 diabetes is associated with a decrease in bone mass and delayed healing of fractures in human and in animal models of type 1 diabetes. Using well- and poorly compensated diabetic BB/O(ttawa) K(arlsburg) rats spontaneously developing insulin-dependent type 1 diabetes, it was recently shown that, in contrast to all other tissues studied, bone is most influenced by metabolic state and seems to be regulated in a manner different from other organs. Therefore, we studied the expression of additional genes (Bmp-1, Bmp-4, Vegf, Bglap, Il-1b, Infg, Tnfa, Calca, Sp1, Yy1) in bone of nondiabetic BB rats compared with newly diagnosed and well- and poorly compensated diabetic rats as well as two nondiabetes-prone congenic BB.SHR rats, BB rat-related (WOKW) and -unrelated rat strains (F344). Six males of each group were euthanized, the tibial bone was removed, and total RNA was extracted, transcribed in complementary DNA, and used for real-time PCR. In a comparison of nondiabetic with diabetic groups, the relative gene expression was reduced by >80% in newly diagnosed and in well-compensated diabetic BB/OK rats. The gene expression in poorly compensated rats increased significantly in 7 of 10 genes and was comparable with those of nondiabetic BB/OK rats. In a comparison of gene expression between diabetes-prone BB/OK and nondiabetes-prone BB.1K, BB.4S, WOKW, and F344 rats, there were no significant differences between newly diagnosed and well-compensated BB/OK diabetic rats and nondiabetic BB.1K, BB.4S, WOKW, and F344 rats. On the basis of these findings, we concluded that spontaneous diabetes influences bone gene expression in BB/OK rats, which may be attributed to the genetically determined autoimmune process not only affecting pancreatic beta-cells but also bone formation and resorption.

Influence of diabetic metabolic state on fracture healing in spontaneously diabetic rats.

BACKGROUND: Type 1 diabetes mellitus (DM) has been shown to alter the properties of bone and impair fracture healing in both humans and animals. The objective of this study was to examine changes in the histomorphometrical, histological and mechanical parameters of bone and remodeling during fracture healing, depending on the diabetic metabolic state in spontaneously diabetic BB/O(ttawa)K(arlsburg) rats, a rat strain that represents a close homology to DM in man. METHODS: On the basis of blood glucose values at the time of surgery, postoperative blood glucose course and postoperative insulin requirements, 90 spontaneously diabetic BB/OK rats were divided into groups with well-compensated or poorly compensated metabolic state. Forty-five LEW.1A rats served as the normoglycemic controls. The femurs were fractured in a standardized procedure and then allowed to heal for 2, 4 and 6 weeks. RESULTS: Our study showed that, in terms of bone histomorphometry, within the first 4 weeks after fracture, severe mineralization disorders occurred exclusively in the rats with poorly compensated diabetic metabolic states with a significant decrease of all fluorochrome-based parameters of mineralization, apposition, formation and timing of mineralization, as well as a delay in cellular differentiation and significantly decreased values of biomechanical properties in comparison to the spontaneously diabetic rats with well-compensated metabolic states and to the control rats. CONCLUSIONS: With a controlled insulin therapy and a resulting well-compensated diabetic metabolic state, mineralization and cellular differentiation disorders, as well as the decreased values of biomechanical properties in the fracture repair in the poorly compensated diabetic metabolic state with very severe hyperglycemia can be avoided.

Diabetes per se and metabolic state influence gene expression in tissue-dependent manner of BB/OK rats.

BACKGROUND: Several epidemiologic studies have clearly established that long-term near normoglycaemia strongly protects against onset and progression of late complication of diabetes. Therefore, insulin treatment plays a crucial role in determining the quality of life of affected individuals. Here we studied the effects of exogenous insulin on gene expression levels in well- and poorly compensated diabetic subjects in comparison to non-diabetic BB/OK rats to find out whether diabetes per se and the quality of insulin treatment have an effect on gene expression and whether it is tissue specific. METHODS: Six non-diabetic and 12 diabetic BB/OK rats were studied. Diabetic subjects were either treated with insulin implants (well compensated) or treated with 1U insulin daily (poorly compensated) to guarantee survival. Four weeks after onset of diabetes, the animals were killed and expression of Yy1, Ppargamma, Nfkappab, Pref-1, Tgfb1, Il-10, and Lepr was measured in thymus, spleen, liver, heart, and bone. RESULTS: In general, between diabetic and non-diabetic subjects, significant expression changes were detected in spleen for Il-10, in heart for Il-10 and Ppargamma, in liver for Yy1, Nfkappab, and Lepr, as well as in bone for all genes studied except Tgfb1. Except Lepr, no expression changes were observed in thymus. Between well- and poorly compensated rats, significant differences on expression level were found for Yy1 (liver), Ppargamma (heart), Nfkappab (bone), Pref-1 (spleen), and Lepr (thymus, liver, heart). CONCLUSION: The insulin treatment compensates not only metabolic disturbances but also changes gene expression profile in BB/OK rats in a tissue-dependent manner.

Histomorphometric evaluation of the influence of the diabetic metabolic state on bone defect healing depending on the defect size in spontaneously diabetic BB/OK rats.

Insulin-dependent type 1 diabetes mellitus (IDDM) has been shown to alter the properties of bone and impair bone repair in both humans and animals. The objective of this study was the detailed histomorphometric evaluation of the influence of the diabetic metabolic state on bone formation and remodeling during bone defect healing depending on the defect size in spontaneously diabetic BB/O(ttawa)K(arlsburg) rats, a rat strain that represents a close homology to IDDM in man. Based on blood-glucose values at the time of surgery, postoperative blood-glucose course, and postoperative insulin requirements, 80 spontaneously diabetic BB/OK rats were divided into groups with well-compensated or poorly compensated metabolic state. Forty LEW.1A rats served as normoglycemic controls. Using a Kirschner wire, bone defects of different sizes were created proximal to the knee joint space in both femora. Ten animals from each group were killed on postoperative days 7, 14, 24, and 42, and specimens were processed undecalcified for quantitative bone histomorphometry. In terms of bone histomorphometry, our study did not show any differences in bone defect healing between the groups where the defect size was 0.4 mm. Larger bone defects (0.8 mm) only showed significant differences in the structural calculations after the 24th postoperative day exclusively in poorly compensated diabetic rats compared to well-compensated diabetic and control rats (P < 0.05 or P < 0.01). In bone defect sizes more than 1.2 mm, severe mineralization disorders occurred within the first 14 days exclusively in rats with poorly compensated diabetic metabolic state with a highly significant (P < 0.001) or significant (P < 0.01) decrease of all fluorochrome-based parameters of mineralization, apposition, formation, and timing of mineralization in comparison to spontaneously diabetic rats with well-compensated diabetic metabolic state and control rats. These results demonstrate that the bone repair of minor bone defects (0.4 mm) is independent of the diabetic metabolic state in spontaneously diabetic BB/OK rats. In larger bone defects (more than 0.8 mm), the bone defect healing in spontaneously diabetic BB/OK rats is impaired exclusively in poorly compensated diabetic metabolic states. This study suggests that strictly controlled insulin treatment resulting in a well-compensated diabetic metabolic state will ameliorate the impaired histomorphometric parameters of IDDM bone defect healing.

Two-year results after lumbar microdiscectomy with and without prophylaxis of a peridural fibrosis using Adcon-L.

BACKGROUND: A retrospective study investigated the advantages of the intraoperative application of the anti-adhesion barrier gel Adcon-L after lumbar microdiscectomy. METHODS: For the study, 92 patients were recruited. Following the operation, 46 patients (study group) received Adcon-L application. In other 46 patients (control group), wounds were closed without Adcon-L application. Median follow-up was 32 (range 26-33) months in the study group and 37 (range 32-41) months in the control group postoperation. All patients were examined clinically, and spinal MRIs were carried out on 12 randomly selected patients in each group. RESULTS: According to the score of the Low Back Pain Committee of the Japanese Orthopedic Association, no significant clinical difference could be established between the two groups. Subjective evaluation of the operation results (score according to Weber) was not significant. Peridural scarring on the postoperative MRI of 24 patients did not differ significantly between the groups. CONCLUSION: A significant effect on the clinical results after Adcon-L application could not be found.

The utility of gait analysis in the rehabilitation of patients after surgical treatment of Achilles tendon rupture.

The purpose of our examinations was to determine whether gait analysis can provide evidence of functional deficits in patients with surgically treated ruptures of the Achilles tendon, and whether measurable parameters for rehabilitation result. In a retrospective study, we examined 30 patients with traumatic Achilles tendon rupture who had been surgically treated immediately posttraumatically with direct tendon suturing between May 1996 and November 1999. We used gait analysis and applied the Hannover Achilles tendon score with regard to occupational and athletic disability. The average follow-up examination interval was 24 (range 18-28) months. After processing results and placing patients in deficit-oriented therapy, a final assessment was conducted using the score mentioned above and a gait analysis. Prior to beginning deficit-oriented therapy, the 100-point score showed that, after an Achilles tendon rupture, 66.7% of the patients had a good to very good outcome. Gait analysis indicated significant deficits in functional mobility during the swing phase, manifesting as a diminishment of the active heel lift. At the time of the final assessment, the findings from gait analysis demonstrated that these deficits were no longer evident to any significant extent. The 100-point score from the final assessment yielded an increase in good and very good results to a total of 80.0%. Gait analysis is suited to both the detection of functional deficits following reconstructive surgery on the musculoskeletal system and to devising individual, patient-oriented rehabilitation plans.