Vaginal distention rodent model for fecal incontinence: a pilot study on the effect on defecation behavior

Objectives: Vaginal balloon inflation simulates the compressive forces on the pelvic floor during the second phase of natural delivery. The foremost use of this animal model of vaginal distention (VD) is to study the mechanisms underlying urinary incontinence. As damage to the pelvic floor during natural birth is a common cause of fecal incontinence, the present paper aimed to investigate the effect of VD on defecation behavior in adult rats. Methods: Vaginal distention was performed in 8 rats for 2 hours, and in 3 rats for 4 hours, and sham inflation was performed in 4 rats. With the use of a latrine box in the rat home-cage and 24/7 video tracking, the defecation behavior was examined. The time spent in and outside the latrine was monitored for two weeks preoperatively and three weeks postoperatively, and a defecation behavior index (DBI; range: 0 [continent] to 1 [incontinent]) was defined. Pelvic floor tissue was collected postmortem and stained with hematoxylin and eosin. Results: Vaginal balloon inflation for 2 hours resulted in fecal incontinence in 29% of the animals (responders) whereas the DBI scores of non-responders (71%) and control animals did not change in the postoperative phase compared with the baseline score. A 4-hour balloon inflation resulted in fecal incontinence in 1 animal and caused a humane endpoint in 2 animals with markedly more tissue damage in the 4-hour responder compared with the 2-hour responders. Conclusions: Vaginal balloon inflation, with an optimum duration between 2 and 4 hours, can be used as a model to study changes in defecation behavior in rats induced by pelvic floor damage. (AU)

Conventional Dorsal Root Ganglion Stimulation in an Experimental Model of Painful Diabetic Peripheral Neuropathy: A Quantitative Immunocytochemical Analysis of Intracellular γ-Aminobutyric Acid in Dorsal Root Ganglion Neurons.

BACKGROUND AND OBJECTIVE: The sensory cell somata in the DRG contain all equipment necessary for extensive GABAergic signaling and are able to release GABA upon depolarization. With this study, we hypothesize that pain relief induced by conventional dorsal root ganglion stimulation (Con-DRGS) in animals with experimental painful diabetic peripheral neuropathy is related to the release of GABA from DRG neurons. With use of quantitative immunocytochemistry, we hypothesize DRGS to result in a decreased intensity of intracellular GABA-immunostaining in DRG somata. MATERIALS AND METHODS: Female Sprague-Dawley rats (n = 31) were injected with streptozotocin (STZ) in order to induce Diabetes Mellitus. Animals that developed neuropathic pain after four weeks (Von Frey) were implanted with a unilateral DRGS device at L4 (n = 14). Animals were then stimulated for 30 min with Con-DRGS (20 Hz, pulse width = 0.2 msec, amplitude = 67% of motor threshold, n = 8) or Sham-DRGS (n = 6), while pain behavior (von Frey) was measured. DRGs were then collected and immunostained for GABA, and a relation to size of sensory cell soma diameter (small: 12-26 µm, assumed to be C-fiber related sensory neurons; medium: 26-40 µm, assumed to be Aδ related sensory neurons; and large: 40-54 µm, assumed to be Aß related sensory neurons) was made. RESULTS: DRGS treated animals showed significant reductions in STZ-induced mechanical hypersensitivity. No significant differences in GABA immunostaining intensity per sensory neuron cell soma type (small-, medium-, or large-sized) were noted in DRGs of stimulated (Con-DRGS) animals versus Sham animals. No differences in GABA immunostaining intensity per sensory cell soma type in ipsi- as compared to contralateral DRGs were observed. CONCLUSION: Con-DRGS does not affect the average intracellular GABA immunofluorescence staining intensity in DRG sensory neurons of those animals which showed significant pain reduction. Similarly, no soma size related changes in intracellular GABA immunofluorescence were observed following Con-DRGS.

Mechanism of dorsal root ganglion stimulation for pain relief in painful diabetic polyneuropathy is not dependent on GABA release in the dorsal horn of the spinal cord.

AIMS: It is hypothesized that dorsal root ganglion stimulation (DRGS), sharing some of the mechanisms of traditional spinal cord stimulation (SCS) of the dorsal columns, induces γ-aminobutyric acid (GABA) release from interneurons in the spinal dorsal horn. METHODS: We used quantitative immunohistochemical analysis in order to investigate the effect of DRGS on intensity of intracellular GABA-staining levels in the L4-L6 spinal dorsal horn of painful diabetic polyneuropathy (PDPN) animals. To establish the maximal pain relieving effect, we tested for mechanical hypersensitivity to von Frey filaments and animals received 30 minutes of DRGS at day 3 after implantation of the electrode. One day later, 4 Sham-DRGS animals and four responders-to-DRGS received again 30 minutes of DRGS and were perfused at the peak of DRGS-induced pain relief. RESULTS: No significant difference in GABA-immunoreactivity was observed between DRGS and Sham-DRGS in lamina 1-3 of the spinal levels L4-6 neither ipsilaterally nor contralaterally. CONCLUSIONS: Dorsal root ganglion stimulation does not induce GABA release from the spinal dorsal horn cells, suggesting that the mechanisms underlying DRGS in pain relief are different from those of conventional SCS. The modulation of a GABA-mediated "Gate Control" in the DRG itself, functioning as a prime Gate of nociception, is suggested and discussed.

Dorsal Root Ganglion Stimulation in Experimental Painful Diabetic Polyneuropathy: Delayed Wash-Out of Pain Relief After Low-Frequency (1Hz) Stimulation.

OBJECTIVE: Up until now there is little data about the pain relieving effect of different frequency settings in DRGS. The aim of this study was to compare the pain relieving effect of DRGS at low-, mid-, and high-frequencies and Sham-DRGS in an animal model of painful diabetic neuropathy (PDPN). MATERIAL AND METHODS: Diabetes mellitus was induced by an intraperitoneal injection of streptozotocin in 8-week-old female Sprague-Dawley rats (n = 24; glucose ≥15 mmol/L: n = 20; mechanical hypersensitivity: n = 15). Five weeks later, a DRGS device was implanted at the L5 DRG. Ten animals were included for stimulation, alternating 30 minutes of low (1 Hz)-, mid (20 Hz)-, and high (1000 Hz)-frequencies and Sham-DRGS during four days, with a pulse width of 0.2 msec (average amplitude: 0.19 ± 0.01 mA), using a randomized cross-over design. The effect on mechanical hypersensitivity of the hind paw to von Frey filaments was evaluated. RESULTS: All DRGS frequencies resulted in a complete reversal of mechanical hypersensitivity and "a clinically relevant reduction" was achieved in 70-80% of animals. No significant differences in maximal pain relieving effect were found between the different frequency treatments (p = 0.24). Animals stimulated at 1000 and 20 Hz returned to baseline mechanical hypersensitivity values 15 and 30 min after stimulation cessation, respectively, while animals stimulated at 1 Hz did not. CONCLUSIONS: These results show that DRGS is equally effective when applied at low-, mid-, and high-frequency in an animal model of PDPN. However, low-frequency-(1 Hz)-DRGS resulted in a delayed wash-out effect, which suggests that this is the most optimal frequency for pain therapy in PDPN as compared to mid- and high-frequency.

Nonlinear Relation Between Burst Dorsal Root Ganglion Stimulation Amplitude and Behavioral Outcome in an Experimental Model of Painful Diabetic Peripheral Neuropathy.

BACKGROUND AND OBJECTIVE: Dorsal root ganglion stimulation (DRGS) has recently emerged as a neuromodulation modality in the treatment of chronic neuropathic pain. The objective of this study was to compare the efficacy of different Burst-DRGS amplitudes in an experimental model of painful diabetic peripheral neuropathy (PDPN). METHODS: Diabetes mellitus was induced in female Sprague-Dawley rats by intraperitoneal injection of streptozotocin (STZ, n = 28). Animals were tested for mechanical hypersensitivity (von Frey paw withdrawal test) before, and four weeks after STZ injection. PDPN rats (n = 13) were implanted with a unilateral bipolar electrode at the L5 DRG. Animals received Burst-DRGS at 0%, 10%, 33%, 50%, 66%, and 80% of motor threshold (MT) in a randomized crossover design on post-implantation days 2-7 (n = 9). Mechanical hypersensitivity was assessed before stimulation onset, 15 and 30 min during stimulation, and 15 and 30 min after stimulation. RESULTS: Burst-DRGS at amplitudes of 33%, 50%, 66%, and 80% MT resulted in significant attenuation of STZ-induced mechanical hypersensitivity at 15 and 30 min during stimulation, as well as 15 min after cessation of stimulation. No effect on mechanical hypersensitivity was observed for Burst-DRGS at 0% MT and 10% MT. Optimal pain relief and highest responder rates were achieved with Burst-DRGS at 50-66% MT, with an estimated optimum at 52% MT. CONCLUSION: Our findings indicate a nonlinear relationship between Burst-DRGS amplitude and behavioral outcome, with an estimated optimal amplitude of 52% MT. Further optimization and analysis of DRGS driven by insights into the underlying mechanisms related to the various stimulation paradigms is warranted.

The Beneficial Effects of UM206 on Wound Healing After Myocardial Infarction in Mice Are Lost in Follow-Up Experiments.

Introduction: An inadequate wound healing following myocardial infarction (MI) is one of the main etiologies of heart failure (HF) development. Interventions aiming at improving this process may contribute to preserving cardiac function after MI. Our group, as well as others, have demonstrated the crucial role of Wnt/frizzled signaling in post-MI remodeling. In this overview, we provide the results of different studies aimed at confirming an initial study from our group, in which we observed beneficial effects of administration of a peptide fragment of Wnt5a, UM206, on infarct healing in a mouse MI model. Methods: Mice were subjected to permanent left coronary artery ligation, and treated with saline (control) or UM206, administered via osmotic minipumps. Cardiac function was assessed by echocardiography and hemodynamic measurements, while infarct size and myofibroblast content were characterized by (immuno)histochemistry. Results: In total, we performed seven follow-up studies, but we were unable to reproduce the beneficial effects of UM206 on infarct healing in most of them. Variations in dose and timing of UM206 administration, its manufacturer and the genetic background of the mice could not restore the phenotype. An in-depth analysis of the datasets revealed that the absence of effect of UM206 coincided with a lack of adverse cardiac remodeling and HF development in all experimental groups, irrespective of the treatment. Discussion: Irreproducibility of experimental observations is a major issue in biomedical sciences. It can arise from a relatively low number of experimental observations in the original study, a faulty hypothesis or a variation in the experimental model that cannot be controlled. In this case, the lack of adverse cardiac remodeling and lung weight increases in the follow-up studies point out to altered experimental conditions as the most likely explanation.

Dorsal Root Ganglion Stimulation in Experimental Painful Diabetic Peripheral Neuropathy: Burst vs. Conventional Stimulation Paradigm.

OBJECTIVES: Painful diabetic peripheral neuropathy (PDPN) is a long-term complication of diabetes mellitus (DM). Dorsal Root Ganglion Stimulation (DRGS) has recently emerged as a neuromodulation modality in the treatment of chronic neuropathic pain. The objective of this study was to compare the effect of burst DRGS (Burst-DRGS) and conventional DRGS (Con-DRGS) in an experimental model of PDPN. MATERIALS AND METHODS: DM was induced in female Sprague-Dawley rats by intraperitoneal injection of streptozotocin (STZ, n = 48). Animals were tested for mechanical hypersensitivity (50% hind paw withdrawal threshold on Von Frey test) before, and 4 weeks after STZ injection. PDPN rats were then implanted with a unilateral bipolar lead at the L5 DRG (n = 22) and were stimulated for 30 min at days 2 and 3 postimplantation. Animals received Con-DRGS and Burst-DRGS in a randomized crossover design (n = 10), or received Sham-DRGS (n = 7) for 30 min, and were tested for mechanical hypersensitivity at baseline, 15 and 30 min during DRGS, and 15 and 30 min following DRGS. Five animals were withdrawn from the study due to electrode-related technical problems. RESULTS: Con-DRGS and Burst-DRGS normalized STZ-induced mechanical hypersensitivity at 15 and 30 min during stimulation. A significant difference in terms of mechanical hypersensitivity was observed between both of the stimulated groups and the Sham-DRGS group at 15 and 30 min during stimulation. Interestingly, Burst-DRGS showed signs of a residual effect at 15 min after cessation of stimulation, while this was not the case for Con-DRGS. CONCLUSIONS: Under the conditions tested, Con-DRGS and Burst-DRGS are equally effective in attenuating STZ-induced mechanical hypersensitivity in an animal model of PDPN. Burst-DRGS showed signs of a residual effect at 15 min after cessation of stimulation, which requires further investigation.

Effectiveness of dorsal root ganglion stimulation and dorsal column spinal cord stimulation in a model of experimental painful diabetic polyneuropathy.

AIMS: Conventional dorsal root ganglion stimulation (DRGS) is known to achieve better pain-paresthesia overlap of difficult-to-reach areas like the feet compared to dorsal column spinal cord stimulation (SCS). As in painful diabetic polyneuropathy (PDPN) pain is mostly present in the feet, we hypothesized that DRGS is more effective in relieving pain in PDPN when compared to SCS. METHODS: Diabetes was induced in female Sprague-Dawley rats with an intraperitoneal injection of 65 mg/kg of streptozotocin (STZ; n = 48). Rats with a significant decrease in mechanical paw withdrawal response to von Frey filaments 4 weeks after injection were implanted with DRGS electrodes (n = 18). Rats were assigned to DRGS (n = 11) or sham-DRGS (n = 7). Mechanical paw withdrawal thresholds (WT, measured in grams) in response to DRGS (50 Hz, 0.18 ± 0.05 mA) were assessed with von Frey testing. The results of the experiments on these animals were compared to the results of a previous study using exactly the same model on PDPN animals selected for SCS (n = 8) (40-50 Hz, 0.19 ± 0.01 mA) and sham-SCS (n = 3). RESULTS: In the SCS group, the log10 (10 000 × 50% WT) increased from 4910 to 5211 at t = 15 minutes (P < 0.05) and 5264 at t = 30 minutes (P = 0.11). In the DRGS group, the log10 (10,000 × 50% WT) increased from 4376 to 4809 at t = 15 minutes (P < 0.01) and 5042 at t = 30 minutes (P < 0.01). Both DRGS and SCS induced a similar and complete reversal of mechanical hypersensitivity. After cessation of stimulation (t = 60), the return of the log10 (10 000 × 50% WT) response was significantly faster with DRGS than that of SCS (P < 0.05). CONCLUSIONS: We conclude that conventional DRGS is as effective as SCS in reduction of PDPN-associated mechanical hypersensitivity in STZ-induced diabetic rats. The wash-in effect of DRGS and SCS was similar, but DRGS showed a faster washout course. Long-term efficacy should be studied in future animal research.

Macrophage Stimulating Protein Enhances Hepatic Inflammation in a NASH Model.

Non-alcoholic steatohepatitis (NASH) is a common liver disease characterized by hepatic lipid accumulation (steatosis) and inflammation. Currently, therapeutic options are poor and the long-term burden to society is constantly increasing. Previously, macrophage stimulating protein (MSP)-a serum protein mainly secreted by liver-was shown to inhibit oxidized low-density lipoprotein (OxLDL)/lipopolysaccharides (LPS)-induced inflammation in mouse macrophages. Additionally, MSP could reduce palmitic acid (PA)-induced lipid accumulation and lipogenesis in the HepG2 cell line. Altogether, these data suggest MSP as a suppressor for metabolic inflammation. However, so far the potential of MSP to be used as a treatment for NASH was not investigated. We hypothesized that MSP reduces lipid accumulation and hepatic inflammation. To investigate the effects of MSP in the early stage of NASH, low-density lipoprotein receptor (Ldlr-/-) mice were fed either a regular chow or a high fat, high cholesterol (HFC) diet for 7 days. Recombinant MSP or saline (control) was administrated to the mice by utilizing subcutaneously-implanted osmotic mini-pumps for the last 4 days. As expected, mice fed an HFC diet showed increased plasma and hepatic lipid accumulation, as well as enhanced hepatic inflammation, compared with chow-fed controls. Upon MSP administration, the rise in cholesterol and triglyceride levels after an HFC diet remained unaltered. Surprisingly, while hepatic macrophage and neutrophil infiltration was similar between the groups, MSP-treated mice showed increased gene expression of pro-inflammatory and pro-apoptotic mediators in the liver, compared with saline-treated controls. Contrary to our expectations, MSP did not ameliorate NASH. Observed changes in inflammatory gene expression suggest that further research is needed to clarify the long-term effects of MSP.

Impaired flow-induced arterial remodeling in DOCA-salt hypertensive rats.

Arteries from young healthy animals respond to chronic changes in blood flow and blood pressure by structural remodeling. We tested whether the ability to respond to decreased (-90%) or increased (+100%) blood flow is impaired during the development of deoxycorticosterone acetate (DOCA)-salt hypertension in rats, a model for an upregulated endothelin-1 system. Mesenteric small arteries (MrA) were exposed to low blood flow (LF) or high blood flow (HF) for 4 or 7 weeks. The bioavailability of vasoactive peptides was modified by chronic treatment of the rats with the dual neutral endopeptidase (NEP)/endothelin-converting enzyme (ECE) inhibitor SOL1. After 3 or 6 weeks of hypertension, the MrA showed hypertrophic arterial remodeling (3 weeks: media cross-sectional area (mCSA): 10±1 × 10(3) to 17±2 × 10(3) µm(2); 6 weeks: 13±2 × 10(3) to 24±3 × 10(3) µm(2)). After 3, but not 6, weeks of hypertension, the arterial diameter was increased (Ø: 385±13 to 463±14 µm). SOL1 reduced hypertrophy after 3 weeks of hypertension (mCSA: 6 × 10(3)±1 × 10(3) µm(2)). The diameter of the HF arteries of normotensive rats increased (Ø: 463±22 µm) but no expansion occurred in the HF arteries of hypertensive rats (Ø: 471±16 µm). MrA from SOL1-treated hypertensive rats did show a significant diameter increase (Ø: 419±13 to 475±16 µm). Arteries exposed to LF showed inward remodeling in normotensive and hypertensive rats (mean Ø between 235 and 290 µm), and infiltration of monocyte/macrophages. SOL1 treatment did not affect the arterial diameter of LF arteries but reduced the infiltration of monocyte/macrophages. We show for the first time that flow-induced remodeling is impaired during the development of DOCA-salt hypertension and that this can be prevented by chronic NEP/ECE inhibition.

Hypertension is associated with marked alterations in sphingolipid biology: a potential role for ceramide.

BACKGROUND: Hypertension is, amongst others, characterized by endothelial dysfunction and vascular remodeling. As sphingolipids have been implicated in both the regulation of vascular contractility and growth, we investigated whether sphingolipid biology is altered in hypertension and whether this is reflected in altered vascular function. METHODS AND FINDINGS: In isolated carotid arteries from spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats, shifting the ceramide/S1P ratio towards ceramide dominance by administration of a sphingosine kinase inhibitor (dimethylsphingosine) or exogenous application of sphingomyelinase, induced marked endothelium-dependent contractions in SHR vessels (DMS: 1.4±0.4 and SMase: 2.1±0.1 mN/mm; n = 10), that were virtually absent in WKY vessels (DMS: 0.0±0.0 and SMase: 0.6±0.1 mN/mm; n = 9, p<0.05). Imaging mass spectrometry and immunohistochemistry indicated that these contractions were most likely mediated by ceramide and dependent on iPLA(2), cyclooxygenase-1 and thromboxane synthase. Expression levels of these enzymes were higher in SHR vessels. In concurrence, infusion of dimethylsphingosine caused a marked rise in blood pressure in anesthetized SHR (42±4%; n = 7), but not in WKY (-12±10%; n = 6). Lipidomics analysis by mass spectrometry, revealed elevated levels of ceramide in arterial tissue of SHR compared to WKY (691±42 vs. 419±27 pmol, n = 3-5 respectively, p<0.05). These pronounced alterations in SHR sphingolipid biology are also reflected in increased plasma ceramide levels (513±19 pmol WKY vs. 645±25 pmol SHR, n = 6-12, p<0.05). Interestingly, we observed similar increases in ceramide levels (correlating with hypertension grade) in plasma from humans with essential hypertension (185±8 pmol vs. 252±23 pmol; n = 18 normotensive vs. n = 19 hypertensive patients, p<0.05). CONCLUSIONS: Hypertension is associated with marked alterations in vascular sphingolipid biology such as elevated ceramide levels and signaling, that contribute to increased vascular tone.

Absence of SPARC results in increased cardiac rupture and dysfunction after acute myocardial infarction.

The matricellular protein SPARC (secreted protein, acidic and rich in cysteine, also known as osteonectin) mediates cell-matrix interactions during wound healing and regulates the production and/or assembly of the extracellular matrix (ECM). This study investigated whether SPARC functions in infarct healing and ECM maturation after myocardial infarction (MI). In comparison with wild-type (WT) mice, animals with a targeted inactivation of SPARC exhibited a fourfold increase in mortality that resulted from an increased incidence of cardiac rupture and failure after MI. SPARC-null infarcts had a disorganized granulation tissue and immature collagenous ECM. In contrast, adenoviral overexpression of SPARC in WT mice improved the collagen maturation and prevented cardiac dilatation and dysfunction after MI. In cardiac fibroblasts in vitro, reduction of SPARC by short hairpin RNA attenuated transforming growth factor beta (TGF)-mediated increase of Smad2 phosphorylation, whereas addition of recombinant SPARC increased Smad2 phosphorylation concordant with increased Smad2 phosphorylation in SPARC-treated mice. Importantly, infusion of TGF-beta rescued cardiac rupture in SPARC-null mice but did not significantly alter infarct healing in WT mice. These findings indicate that local production of SPARC is essential for maintenance of the integrity of cardiac ECM after MI. The protective effects of SPARC emphasize the potential therapeutic applications of this protein to prevent cardiac dilatation and dysfunction after MI.

Lysosomal integral membrane protein 2 is a novel component of the cardiac intercalated disc and vital for load-induced cardiac myocyte hypertrophy.

The intercalated disc (ID) of cardiac myocytes is emerging as a crucial structure in the heart. Loss of ID proteins like N-cadherin causes lethal cardiac abnormalities, and mutations in ID proteins cause human cardiomyopathy. A comprehensive screen for novel mechanisms in failing hearts demonstrated that expression of the lysosomal integral membrane protein 2 (LIMP-2) is increased in cardiac hypertrophy and heart failure in both rat and human myocardium. Complete loss of LIMP-2 in genetically engineered mice did not affect cardiac development; however, these LIMP-2 null mice failed to mount a hypertrophic response to increased blood pressure but developed cardiomyopathy. Disturbed cadherin localization in these hearts suggested that LIMP-2 has important functions outside lysosomes. Indeed, we also find LIMP-2 in the ID, where it associates with cadherin. RNAi-mediated knockdown of LIMP-2 decreases the binding of phosphorylated beta-catenin to cadherin, whereas overexpression of LIMP-2 has the opposite effect. Collectively, our data show that LIMP-2 is crucial to mount the adaptive hypertrophic response to cardiac loading. We demonstrate a novel role for LIMP-2 as an important mediator of the ID.

Biologically relevant effects of mRNA amplification on gene expression profiles.

BACKGROUND: Gene expression microarray technology permits the analysis of global gene expression profiles. The amount of sample needed limits the use of small excision biopsies and/or needle biopsies from human or animal tissues. Linear amplification techniques have been developed to increase the amount of sample derived cDNA. These amplified samples can be hybridised on microarrays. However, little information is available whether microarrays based on amplified and unamplified material yield comparable results. In the present study we compared microarray data obtained from amplified mRNA derived from biopsies of rat cardiac left ventricle and non-amplified mRNA derived from the same organ. Biopsies were linearly amplified to acquire enough material for a microarray experiment. Both amplified and unamplified samples were hybridized to the Rat Expression Set 230 Array of Affymetrix. RESULTS: Analysis of the microarray data showed that unamplified material of two different left ventricles had 99.6% identical gene expression. Gene expression patterns of two biopsies obtained from the same parental organ were 96.3% identical. Similarly, gene expression pattern of two biopsies from dissimilar organs were 92.8% identical to each other.Twenty-one percent of reporters called present in parental left ventricular tissue disappeared after amplification in the biopsies. Those reporters were predominantly seen in the low intensity range. Sequence analysis showed that reporters that disappeared after amplification had a GC-content of 53.7+/-4.0%, while reporters called present in biopsy- and whole LV-samples had an average GC content of 47.8+/-5.5% (P <0.001). Those reporters were also predicted to form significantly more (0.76+/-0.07 versus 0.38+/-0.1) and longer (9.4+/-0.3 versus 8.4+/-0.4) hairpins as compared to representative control reporters present before and after amplification. CONCLUSION: This study establishes that the gene expression profile obtained after amplification of mRNA of left ventricular biopsies is representative for the whole left ventricle of the rat heart. However, specific gene transcripts present in parental tissues were undetectable in the minute left ventricular biopsies. Transcripts that were lost due to the amplification process were not randomly distributed, but had higher GC-content and hairpins in the sequence and were mainly found in the lower intensity range which includes many transcription factors from specific signalling pathways.

Long-term structural and functional consequences of cardiac ischaemia-reperfusion injury in vivo in mice.

The short-term (<24 h) consequences of oxidative stress induced by ischaemia-reperfusion (IR) have been studied extensively in the mouse heart. However, much less is known about the long-term effects inflicted by a brief ischaemic period on the murine heart. We therefore examined the structural and functional consequences of a 30 min ischaemic period after 2 and 8 weeks of reperfusion and compared these to the effects induced by permanent occlusion of the left anterior descending coronary artery (LAD). The latter procedure resulted in transmural myocardial infarcts of about 52% of the left ventricle. In contrast, the single 30 min ischaemic period led to infarct sizes of about 13% of the left ventricle (range, 4-23%) at 2 and 8 weeks after reperfusion. Maximal cardiac contractility responses (+dP/dt) to dobutamine infusion and volume loading were depressed at 2, but not at 8 weeks after IR. The restoration of cardiac contractility at 8 weeks after IR was associated with a significant 20% enlargement of the end-diastolic volume and 16% increase of the left ventricular wall thickness. These changes in cardiac geometry were less pronounced at 2 weeks after IR. Histological examination revealed that the IR injury was associated with prominent calcification. At 2 and at 8 weeks after IR, 25 +/- 5 and 38 +/- 5% of the injured area was calcified as observed in 69 and 73% of the animals, respectively. After permanent occlusion of the LAD, calcification was not observed and healing of the affected area was characterized by thinning and dilatation of the infarcted myocardium. These data indicate that, in mice, a single 30 min period of ischaemia reduced ventricular contractility up to at least 2 weeks after reperfusion. However, 8 weeks after IR, cardiac function was restored by eccentric hypertrophy associated with calcification of the injured ventricular wall.

Thrombospondin-2 is essential for myocardial matrix integrity: increased expression identifies failure-prone cardiac hypertrophy.

Cardiac hypertrophy can lead to heart failure (HF), but it is unpredictable which hypertrophied myocardium will progress to HF. We surmised that apart from hypertrophy-related genes, failure-related genes are expressed before the onset of failure, permitting molecular prediction of HF. Hearts from hypertensive homozygous renin-overexpressing (Ren-2) rats that had progressed to early HF were compared by microarray analysis to Ren-2 rats that had remained compensated. To identify which HF-related genes preceded failure, cardiac biopsy specimens were taken during compensated hypertrophy and we then monitored whether the rat progressed to HF or remained compensated. Among 48 genes overexpressed in failing hearts, we focused on thrombospondin-2 (TSP2). TSP2 was selectively overexpressed only in biopsy specimens from rats that later progressed to HF. Moreover, expression of TSP2 was increased in human hypertrophied hearts with decreased (0.19+/-0.01) versus normal ejection fraction (0.11+/-0.03 [arbitrary units]; P<0.05). Angiotensin II induced fatal cardiac rupture in 70% of TSP2 knockout mice, with cardiac failure in the surviving mice; this was not seen in wild-type mice. In TSP2 knockout mice, angiotensin II increased matrix metalloproteinase (MMP)-2 and MMP-9 activity by 120% and 390% compared with wild-type mice (P<0.05). In conclusion, we identify TSP2 as a crucial regulator of the integrity of the cardiac matrix that is necessary for the myocardium to cope with increased loading and that may function by its regulation of MMP activity. This suggests that expression of TSP2 marks an early-stage molecular program that is activated uniquely in hypertrophied hearts that are prone to fail.

Effects of anesthetics on systemic hemodynamics in mice.

The aim of this study was to compare the systemic hemodynamic effects of four commonly used anesthetic regimens in mice that were chronically instrumented for direct and continuous measurements of cardiac output (CO). Mice (CD-1, Swiss, and C57BL6 strains) were instrumented with a transit-time flow probe placed around the ascending aorta for CO measurement. An arterial catheter was inserted into the aorta 4 or 5 days later for blood pressure measurements. After full recovery, hemodynamic parameters including stroke volume, heart rate, CO, mean arterial pressure (MAP), and total peripheral resistance were measured with animals in the conscious state. General anesthesia was then induced in these mice using isoflurane (Iso), urethane, pentobarbital sodium, or ketamine-xylazine (K-X). The doses and routes of administration of these agents were given as required for general surgical procedures in these animals. Compared with the values obtained for animals in the conscious resting state, MAP and CO decreased during all anesthetic interventions, and hemodynamic effects were smallest for Iso (MAP, -24 +/- 3%; CO, -5 +/- 7%; n = 15 mice) and greatest for K-X (MAP, -51 +/- 6%; CO, -37 +/- 9%; n = 8 mice), respectively. The hemodynamic effects of K-X were fully antagonized by administration of the alpha(2)-receptor antagonist atipamezole (n = 8 mice). These results indicate that the anesthetic Iso has fewer systemic hemodynamic effects in mice than the nonvolatile anesthetics.