Factors affecting 39K NMR detectability in rat tissue

In this study we have found that NMR detectability of 39K in rat thigh muscle may be substantially higher (up to 100% oftotal tissue potassium) than values previously reported of around 40%. The signal was found to consist of two superimposed components, one broad and one narrow, of approximately equal area. Investigations involving improvements in spectral parameters such as signal-to-noise ratio and baseline roll, together with computer simulations of spectra, show that the quality of the spectra has a major effect on the amount of signal detected, which is largely due to the loss of detectability of the broad signal component. In particular, lower-field spectrometers using conventional probes and detection methods generally have poorer signal-to-noise and worse baseline roll artifacts, which make detection of a broad component of the muscle signal difficult.

Effect of magnesium depletion and potassium and chlorothiazide on intracellular pH in the rat, studied by 31P NMR

1. Both dietary magnesium depletion and potassium depletion (confirmed by tissue analysis) were induced in rats which were then compared with rats treated with chlorothiazide (250 mg/kg diet) and rats on a control synthetic diet. 2. Brain and muscle intracellular pH was measured by using a surface coil and [31P]-NMR to measure the chemical shift of inorganic phosphate. pH was also measured in isolated perfused hearts from control and magnesium-deficient rats. Intracellular magnesium status was assessed by measuring the chemical shift of β-ATP in brain. 3. There was no evidence for magnesium deficiency in the chlorothiazide-treated rats on tissue analysis or on chemical shift of β-ATP in brain. Both magnesium and potassium deficiency, but not chlorothiazide treatment, were associated with an extracellular alkalosis. 4. Magnesium deficiency led to an intracellular alkalosis in brain, muscle and heart. Chlorothiazide treatment led to an alkalosis in brain. Potassium deficiency was associated with a normal intracellular pH in brain and muscle. 5. Magnesium depletion and chlorothiazide treatment produce intracellular alkalosis by unknown mechanism(s).

Problems in the assessment of magnesium depletion in the rat by in vivo 31P NMR

Prior in vitro studies, utilizing 31Pn uclear magnetic resonance (31PN MR) to measure the chemical shift (CT) of 0-ATP and lengthening of the phosphocreatine spin-spin (7"') relaxation time, suggested an assessment of their efficacy in measuring magnesium depletion in vivo. Dietary magnesium depletion (Me$) produced markedly lower magnesium in plasma (0.44 vs 1. I3 mmol/liter) and bone (1 30 vs 190 pmol/g) but much smaller changes in muscle (41 vs 45 pmol/g, P < 0.01), heart (42.5 vs 44.6 prnol/g), and brain (30 vs 32 pmollg). NMR experiments in anesthetized rats in a Bruker 7-T vertical bore magnet showed that in M e $ rats there was a significant change in brain j3-ATP shift (16.15 vs 16.03 ppm, P < 0.05). These chemical shifts gave a calculated free [Mg"] of 0.71 mM (control) and 0.48 mM (MgZ+$). In muscle the change in j3-ATP shift was not significant (Me$ 15.99 ppm, controls 15.96 ppm), corresponding to a calculated free M P of 0.83 and 0.95 mM, respectively. Phosphccreatine Tz (Carr-Purcell, spin-echo pulse sequence) was no different with M e $ in muscle in vivo (surface coil) (M$+$ 136, control 142 ms) or in isolated perfused hearts (Helmholtz coil) (control 83, M e $ 92 ms). 3'P NMR is severely limited in its ability to detect dietary magnesium depletion in vivo. Measurement of j3-ATP shift in brain may allow studies of the effects of interaction in group studies but does not allow prediction of an individual magnesium status.

Aromatic l-amino acid decarboxylase : histochemical localization in rat kidney and lack of effect of dietary potassium or sodium loading on enzyme distribution

Utilizing a mono-specific antiserum produced in rabbits to hog kidney aromatic L-amino acid decarboxylase (AADC), the enzyme was localized in rat kidney by immunoperoxidase staining. AADC was located predominantly in the proximal convoluted tubules; there was also weak staining in the distal convoluted tubules and collecting ducts. An increase in dietary potassium or sodium intake produced no change in density or distribution of AADC staining in kidney. An assay of AADC enzyme activity showed no difference in cortex or medulla with chronic potassium loading. A change in distribution or activity of renal AADC does not explain the postulated dopaminergic modulation of renal function that occurs with potassium or sodium loading.

Non-destructive evaluation of articular cartilage defects using near-infrared (NIR) spectroscopy in osteoarthritic rat models and its direct relation to Mankin Score

Objective The aim of this study was to demonstrate the potential of near-infrared (NIR) spectroscopy for categorizing cartilage degeneration induced in animal models. Method Three models of osteoarthritic degeneration were induced in laboratory rats via one of the following methods: (i) menisectomy (MSX); (ii) anterior cruciate ligament transaction (ACLT); and (iii) intra-articular injection of mono-ido-acetete (1 mg) (MIA), in the right knee joint, with 12 rats per model group. After 8 weeks, the animals were sacrificed and tibial knee joints were collected. A custom-made nearinfrared (NIR) probe of diameter 5 mm was placed on the cartilage surface and spectral data were acquired from each specimen in the wavenumber range 4 000 – 12 500 cm−1. Following spectral data acquisition, the specimens were fixed and Safranin–O staining was performed to assess disease severity based on the Mankin scoring system. Using multivariate statistical analysis based on principal component analysis and partial least squares regression, the spectral data were then related to the Mankinscores of the samples tested. Results Mild to severe degenerative cartilage changes were observed in the subject animals. The ACLT models showed mild cartilage degeneration, MSX models moderate, and MIA severe cartilage degenerative changes both morphologically and histologically. Our result demonstrate that NIR spectroscopic information is capable of separating the cartilage samples into different groups relative to the severity of degeneration, with NIR correlating significantly with their Mankinscore (R2 = 88.85%). Conclusion We conclude that NIR is a viable tool for evaluating articularcartilage health and physical properties such as change in thickness with degeneration.

Reversal of acute coagulopathy during hypotensive resuscitation using small-volume 7.5% NaCl adenocaine and Mg2+ in the rat model of severe hemorrhagic shock

OBJECTIVE: : Acute traumatic coagulopathy occurs early in hemorrhagic trauma and is a major contributor to mortality and morbidity. Our aim was to examine the effect of small-volume 7.5% NaCl adenocaine (adenosine and lidocaine, adenocaine) and Mg on hypotensive resuscitation and coagulopathy in the rat model of severe hemorrhagic shock. DESIGN: : Prospective randomized laboratory investigation. SUBJECTS: : A total of 68 male Sprague Dawley Rats. INTERVENTION: : Post-hemorrhagic shock treatment for acute traumatic coagulopathy. MEASUREMENTS AND METHODS: : Nonheparinized male Sprague-Dawley rats (300-450 g, n = 68) were randomly assigned to either: 1) untreated; 2) 7.5% NaCl; 3) 7.5% NaCl adenocaine; 4) 7.5% NaCl Mg; or 5) 7.5% NaCl adenocaine/Mg. Hemorrhagic shock was induced by phlebotomy to mean arterial pressure of 35-40 mm Hg for 20 mins (~40% blood loss), and animals were left in shock for 60 mins. Bolus (0.3 mL) was injected into the femoral vein and hemodynamics monitored. Blood was collected in Na citrate (3.2%) tubes, centrifuged, and the plasma snap frozen in liquid N2 and stored at -80°C. Coagulation was assessed using activated partial thromboplastin times and prothrombin times. RESULTS: : Small-volume 7.5% NaCl adenocaine and 7.5% NaCl adenocaine/Mg were the only two groups that gradually increased mean arterial pressure 1.6-fold from 38-39 mm Hg to 52 and 64 mm Hg, respectively, at 60 mins (p < .05). Baseline plasma activated partial thromboplastin time was 17 ± 0.5 secs and increased to 63 ± 21 secs after bleeding time, and 217 ± 32 secs after 60-min shock. At 60-min resuscitation, activated partial thromboplastin time values for untreated, 7.5% NaCl, 7.5% NaCl/Mg, and 7.5% NaCl adenocaine rats were 269 ± 31 secs, 262 ± 38 secs, 150 ± 43 secs, and 244 ± 38 secs, respectively. In contrast, activated partial thromboplastin time for 7.5% NaCl adenocaine/Mg was 24 ± 2 secs (p < .05). Baseline prothrombin time was 28 ± 0.8 secs (n = 8) and followed a similar pattern of correction. CONCLUSIONS: : Plasma activated partial thromboplastin time and prothrombin time increased over 10-fold during the bleed and shock periods prior to resuscitation, and a small-volume (~1 mL/kg) IV bolus of 7.5% NaCl AL/Mg was the only treatment group that raised mean arterial pressure into the permissive range and returned activated partial thromboplastin time and prothrombin time clotting times to baseline at 60 mins.

Expression and distribution of cell-surface proteoglycans in the normal Lewis rat molar periodontium

Cell-surface proteoglycans participate in several biological functions such as cell cell and cell-matrix interactions, cell adhesion, the binding to various growth factors as co-receptors and repair. To understand better the expression and distribution of cell-surface proteoglycans in the periodontal tissues, an immunohistochemical evaluation of the normal Lewis rat molar periodontium using panels of antibodies for syndecan-1, -2, -4, glypican and betaglycan was carried out. Our results demonstrated the expression and distribution of all proteoglycans in the suprabasal gingival epithelium, soft and hard connective tissues. Both cellular and matrix localization was evident within the various periodontal compartments. The presence of these cell-surface proteoglycans indicates the potential for roles in the process of tissue homeostasis, repair or regeneration in periodontium of which each function requires further study.

Growth-hormone-stimulated dentinogenesis in Lewis dwarf rat molars

In dentinogenesis, certain growth factors, matrix proteoglycans, and proteins are directly or indirectly dependent on growth hormone. The hypothesis that growth hormone up-regulates the expression of enzymes, sialoproteins, and other extracellular matrix proteins implicated in the formation and mineralization of tooth and bone matrices was tested by the treatment of Lewis dwarf rats with growth hormone over 5 days. The molar teeth were processed for immunohistochemical demonstration of bone-alkaline phosphatase, bone morphogenetic proteins-2 and -4, osteocalcin, osteopontin, bone sialoprotein, and E11 protein. Odontoblasts responded to growth hormone by more cells expressing bone morphogenetic protein, alkaline phosphatase, osteocalcin, and osteopontin. No changes were found in bone sialoprotein or E11 protein expression. Thus, growth hormone may stimulate odontoblasts to express several growth factors and matrix proteins associated with dentin matrix biosynthesis in mature rat molars.

The expression of plasminogen activator system in a rat model of periodontal wound healing

BACKGROUND: The plasminogen activator system has been proposed to play a role in proteolytic degradation of extracellular matrices in tissue remodeling, including wound healing. The aim of this study was to elucidate the presence of components of the plasminogen activator system during different stages of periodontal wound healing. METHODS: Periodontal wounds were created around the molars of adult rats and healing was followed for 28 days. Immunohistochemical analyses of the healing tissues and an analysis of the periodontal wound healing fluid by ELISA were carried out for the detection of tissue-type plasminogen activator (t-PA), urokinase-type plasminogen activator (u-PA), and 2 plasminogen activator inhibitors (PAI-1 and PAI-2). RESULTS: During the early stages (days 1 to 3) of periodontal wound healing, PAI-1 and PAI-2 were found to be closely associated with the deposition of a fibrin clot in the gingival sulcus. These components were strongly associated with the infiltrating inflammatory cells around the fibrin clot. During days 3 to 7, u-PA, PAI-1, and PAI-2 were associated with cells (particularly monocytes/macrophages, fibroblasts, and endothelial cells) in the newly formed granulation tissue. During days 7 to 14, a new attachment apparatus was formed during which PAI-1, PAI-2, and u-PA were localized in both periodontal ligament fibroblasts (PDL) and epithelial cells at sites where these cells were attaching to the root surface. In the periodontal wound healing fluid, the concentration for t-PA increased and peaked during the first week. PAI-2 had a similar expression to t-PA, but at a lower level over the entire wound-healing period. CONCLUSIONS: These findings indicate that the plasminogen activator system is involved in the entire process of periodontal wound healing, in particular with the formation of fibrin matrix on the root surface and its replacement by granulation tissue, as well as the subsequent formation of the attachment of soft tissue to the root surface during the later stages of wound repair.

Differential expression and distribution of syndecan-1 and -2 in periodontal wound healing of the rat

Cell-surface proteoglycans participate in several biological functions including interactions with adhesion molecules, growth factors and a variety of other effector molecules. Accordingly, these molecules play a central role in various aspects of cell–cell and cell–matrix interactions. To investigate the expression and distribution of the cell surface proteoglycans, syndecan-1 and -2, during periodontal wound healing, immunohistochemical analyses were carried out using monoclonal antibodies against syndecan-1, or -2 core proteins. Both syndecan-1 and -2 were expressed and distributed differentially at various stages of early inflammatory cell infiltration, granulation tissue formation, and tissue remodeling in periodontal wound healing. Expression of syndecan-1 was noted in inflammatory cells within and around the fibrin clots during the earliest stages of inflammatory cell infiltration. During granulation tissue formation it was noted in fibroblast-like cells and newly formed blood vessels. Syndecan-1 was not seen in newly formed bone or cementum matrix at any of the time periods studied. Syndecan-1 expression was generally less during the late stages of wound healing but was markedly expressed in cells that were close to the repairing junctional epithelium. In contrast, syndecan-2 expression and distribution was not evident at the early stages of inflammatory cell infiltration. During the formation of granulation tissue and subsequent tissue remodeling, syndecan-2 was expressed extracellularly in the newly formed fibrils which were oriented toward the root surface. Syndecan-2 was found to be significantly expressed on cells that were close to the root surface and within the matrix of repaired cementum covering root dentin as well as at the alveolar bone edge. These findings indicate that syndecan-1 and -2 may have distinctive functions during wound healing of the periodontium. The appearance of syndecan-1 may involve both cell–cell and cell–matrix interactions, while syndecan-2 showed a predilection to associate with cell–matrix interactions during hard tissue formation.

Near infrared (NIR) absorption spectra correlates with subchondral bone micro-CT parameters in osteoarthritic rat models

Determining the properties and integrity of subchondral bone in the developmental stages of osteoarthritis, especially in a form that can facilitate real-time characterization for diagnostic and decision-making purposes, is still a matter for research and development. This paper presents relationships between near infrared absorption spectra and properties of subchondral bone obtained from 3 models of osteoarthritic degeneration induced in laboratory rats via: (i) menisectomy (MSX); (ii) anterior cruciate ligament transaction (ACL); and (iii) intra-articular injection of mono-ido-acetate (1 mg) (MIA), in the right knee joint, with 12 rats per model group (N = 36). After 8 weeks, the animals were sacrificed and knee joints were collected. A custom-made diffuse reflectance NIR probe of diameter 5 mm was placed on the tibial surface and spectral data were acquired from each specimen in the wavenumber range 4000–12 500 cm− 1. After spectral acquisition, micro computed tomography (micro-CT) was performed on the samples and subchondral bone parameters namely: bone volume (BV) and bone mineral density (BMD) were extracted from the micro-CT data. Statistical correlation was then conducted between these parameters and regions of the near infrared spectra using multivariate techniques including principal component analysis (PCA), discriminant analysis (DA), and partial least squares (PLS) regression. Statistically significant linear correlations were found between the near infrared absorption spectra and subchondral bone BMD (R2 = 98.84%) and BV (R2 = 97.87%). In conclusion, near infrared spectroscopic probing can be used to detect, qualify and quantify changes in the composition of the subchondral bone, and could potentially assist in distinguishing healthy from OA bone as demonstrated with our laboratory rat models.

Rat meets iRat

Biorobotics has the potential to provide an integrated understanding from neural systems to behavior that is neither ethical nor technically feasible with living systems. Robots that can interact with animals in their natural environment open new possibilities for empirical studies in neuroscience. However, designing a robot that can interact with a rodent requires considerations that span a range of disciplines. For the rat's safety, the body form and movements of the robot need to take into consideration the safety of the animal, an appropriate size for the rodent arenas, and behaviors for interaction. For the robot's safety, its form must be robust in the face of typically inquisitive and potentially aggressive behaviors by the rodent, which can include chewing on exposed parts, including electronics, and deliberate or accidental fouling. We designed a rat-sized robot, the iRat (intelligent rat animat technology) for studies in neuroscience. The iRat is about the same size as a rat and has the ability to navigate autonomously around small environments. In this study we report the first interactions between the iRat and real rodents in a free exploration task. Studies with five rats show that the rats and iRat interact safely for both parties.

Effects of hyperbaric oxygen and inducible nitric oxide synthase inhibitor treatment on femoral head osteonecrosis in a rat model

Introduction: Apoptosis is the final destiny of many cells in the body, though this process has been observed in some pathological processes. One of these pathological processes is femoral head non-traumatic osteonecrosis. Among many pro/anti-apoptotic factors, nitric oxide has recently been an area of further interest. Osteocyte apoptosis and its relation to pro-apoptotic action invite further research, and the inducible form of nitric oxide synthase (iNOS)—which produces a high concentration of nitric oxide—has been flagged. The aim of this study was to investigate the effect of hyperbaric oxygen (HBO) and inducible NOS suppressor (Aminoguanidine) in prevention of femoral head osteonecrosis in an experimental model of osteonecrosis in spontaneous hypertensive rats (SHRs). Methods: After animal ethic approval 34 SHR rats were divided into four groups. Ten rats were allocated to the control group without any treatment, and eight rats were allocated to three treatment groups namely: HBO, Aminoguanidine (AMG), and the combination of HBO and AMG treatments (HBO+AMG). The HBO group received 250 kPa of oxygen via hyperbaric chamber for 30 days started at their 5th week of life; the AMG group received 1mg/ml of AMG in drinking water from the fifth week till the 17th week of life; and the last group received a combination of these treatments. Rats were sacrificed at the end of the 17th week of life and both femurs were analysed for evidence of osteonecrosis using Micro CT scan and H&E staining. Also, osteocyte apoptosis and the presence of two different forms of NOS (inducible (iNOS) and endothelial (eNOS)) were analysed by immunostaining and apoptosis staining (Hoechst and TUNEL). Results: Bone morphology of metaphyseal and epiphyseal area of all rats were investigated and analysed. Micro CT findings revealed significantly higher mean fractional trabecular bone volume (FBV) of metaphyseal area in untreated SHRs compared with all other treatments (HBO, P<0.05, HBO+AMG, P<0.005, and AMG P<0.001). Bone surface to volume ratio also significantly increased with HBO+AMG and AMG treatments when compared with the control group (18.7 Vs 20.8, P<0.05, and 18.7 Vs 21.1, P<0.05). Epiphyseal mean FBV did not change significantly among groups. In the metaphyseal area, trabecular thickness and numbers significantly decreased with AMG treatment, while trabecular separation significantly increased with both AMG and HBO+AMG treatment. Histological ratio of no ossification and osteonecrosis was 37.5%, 43.7%, 18.7% and 6.2% of control, HBO, HBO+AMG and AMG groups respectively with only significant difference observed between HBO and AMG treatment (P<0.01). High concentration of iNOS was observed in the region of osteonecrosis while there was no evidence of eNOS activity around that region. In comparison with the control group, the ratio of osteocyte apoptosis significantly reduced in AMG treatment (P<0.005). We also observed significantly fewer apoptotic osteocytes in AMG group comparing with HBO treatment (P<0.05). Conclusion: None of our treatments prevents osteonecrosis at the histological or micro CT scan level. High concentration of iNOS in the region of osteonecrosis and significant reduction of osteocyte apoptosis with AMG treatment were supportive of iNOS modulating osteocyte apoptosis in SHRs.