GABA-Glycine Cotransmitting Neurons in the Ventrolateral Medulla: Development and Functional Relevance for Breathing.

Inhibitory neurons crucially contribute to shaping the breathing rhythm in the brain stem. These neurons use GABA or glycine as neurotransmitter; or co-release GABA and glycine. However, the developmental relationship between GABAergic, glycinergic and cotransmitting neurons, and the functional relevance of cotransmitting neurons has remained enigmatic. Transgenic mice expressing fluorescent markers or the split-Cre system in inhibitory neurons were developed to track the three different interneuron phenotypes. During late embryonic development, the majority of inhibitory neurons in the ventrolateral medulla are cotransmitting cells, most of which differentiate into GABAergic and glycinergic neurons around birth and around postnatal day 4, respectively. Functional inactivation of cotransmitting neurons revealed an increase of the number of respiratory pauses, the cycle-by-cycle variability, and the overall variability of breathing. In summary, the majority of cotransmitting neurons differentiate into GABAergic or glycinergic neurons within the first 2 weeks after birth and these neurons contribute to fine-tuning of the breathing pattern.

Monitoring ATP dynamics in electrically active white matter tracts.

In several neurodegenerative diseases and myelin disorders, the degeneration profiles of myelinated axons are compatible with underlying energy deficits. However, it is presently impossible to measure selectively axonal ATP levels in the electrically active nervous system. We combined transgenic expression of an ATP-sensor in neurons of mice with confocal FRET imaging and electrophysiological recordings of acutely isolated optic nerves. This allowed us to monitor dynamic changes and activity-dependent axonal ATP homeostasis at the cellular level and in real time. We find that changes in ATP levels correlate well with compound action potentials. However, this correlation is disrupted when metabolism of lactate is inhibited, suggesting that axonal glycolysis products are not sufficient to maintain mitochondrial energy metabolism of electrically active axons. The combined monitoring of cellular ATP and electrical activity is a novel tool to study neuronal and glial energy metabolism in normal physiology and in models of neurodegenerative disorders.

A Transgenic Mouse Line Expressing the Red Fluorescent Protein tdTomato in GABAergic Neurons.

GABAergic inhibitory neurons are a large population of neurons in the central nervous system (CNS) of mammals and crucially contribute to the function of the circuitry of the brain. To identify specific cell types and investigate their functions labelling of cell populations by transgenic expression of fluorescent proteins is a powerful approach. While a number of mouse lines expressing the green fluorescent protein (GFP) in different subpopulations of GABAergic cells are available, GFP expressing mouse lines are not suitable for either crossbreeding to other mouse lines expressing GFP in other cell types or for Ca2+-imaging using the superior green Ca2+-indicator dyes. Therefore, we have generated a novel transgenic mouse line expressing the red fluorescent protein tdTomato in GABAergic neurons using a bacterial artificial chromosome based strategy and inserting the tdTomato open reading frame at the start codon within exon 1 of the GAD2 gene encoding glutamic acid decarboxylase 65 (GAD65). TdTomato expression was observed in all expected brain regions; however, the fluorescence intensity was highest in the olfactory bulb and the striatum. Robust expression was also observed in cortical and hippocampal neurons, Purkinje cells in the cerebellum, amacrine cells in the retina as well as in cells migrating along the rostral migratory stream. In cortex, hippocampus, olfactory bulb and brainstem, 80% to 90% of neurons expressing endogenous GAD65 also expressed the fluorescent protein. Moreover, almost all tdTomato-expressing cells coexpressed GAD65, indicating that indeed only GABAergic neurons are labelled by tdTomato expression. This mouse line with its unique spectral properties for labelling GABAergic neurons will therefore be a valuable new tool for research addressing this fascinating cell type.

Catecholamines can induce pulmonary remodeling in rats.

BACKGROUND/AIMS: Previously, we found that catecholamine (CA) infusion in rats induced pulmonary injury with edema and inflammation resembling acute lung injury in humans. Here, we examined effects of norepinephrine (NE) and of selective α- and ß-adrenergic agonists on the remodeling of pulmonary extracellular matrix. METHODS: Eighty rats were infused over 8-72 h with NE, phenylephrine (PE), isoproterenol (ISO) or NaCl solution. We investigated mRNA expression of collagen, matrix metalloproteinase (MMP)-2, its tissue inhibitor (TIMP-2) and transforming growth factor (TGF)-ß isoforms in lung tissue. Additionally, lung histology, hemodynamic function and cardiac hypertrophy were evaluated. RESULTS: After 72 h of infusion, lung histology showed beginning fibrosis and vascular hypertrophy. Collagen type I, MMP-2 and TIMP-2 mRNA expression were significantly elevated. All these effects were most pronounced with NE while PE and ISO induced weaker responses. TGF-ß mRNA expression was also elevated after 72 h, predominantly after PE infusion. Cardiac hypertrophy was most pronounced after ISO infusion. CONCLUSION: CA infusion over 72 h may induce pulmonary remodeling. Mainly α-adrenergic but also ß-adrenergic mechanisms contribute to these processes. In contrast, cardiac hypertrophy is predominantly mediated by ß-adrenergic stimulation and hence, is considered to be a direct adrenergic effect rather than a consequence of pulmonary fibrosis.

Long-term respiratory muscle endurance training in patients with myasthenia gravis: first results after four months of training.

Myasthenia gravis (MG) is characterized by reduced muscle endurance and is often accompanied by respiratory complications. Improvement of respiratory function is therefore an important objective in MG therapy. A previous study demonstrated that respiratory muscle endurance training (RMET) over four weeks increased respiratory muscle endurance of MG patients to about 200% of baseline. The purpose of the present study was to establish an appropriate maintenance training and to test its effects over four months. Ten patients with mild to moderate MG participated in this study. During the first month, they performed five training sessions per week. For the following 3 months, training frequency was reduced to five sessions per two weeks. Myasthenia score, lung function, and respiratory endurance were determined prior to training, after the first month, and after 4 months. Myasthenia score improved from 0.71 ± 0.1 to 0.56 ± 0.1 (P = 0.007). Respiratory endurance time increased from 6.1 ± 0.8 to 20.3 ± 3.0 min (P < 0.001). In conclusion, this RMET maintenance program is feasible and is significantly beneficial for MG patients.

Crucial role of interleukin-6 in the development of norepinephrine-induced left ventricular remodeling in mice.

BACKGROUND: Elevated serum concentration of interleukin (IL)-6 is a predictor for poor prognosis in congestive heart failure. It was shown previously in rats, that IL-6 expression in the left ventricle (LV) was followed by LV hypertrophy. METHODS: Using IL-6 deficient mice (IL-6(-/-)), we studied the role of IL-6 in a model of norepinephrine (NE)-induced LV hypertrophy. RESULTS: In wild type (WT) mice, IL-6 mRNA expression and its concentration in the serum were elevated after 4 h of NE-treatment (s.c. 0.25 mg.h)./kg Further, NE-induced LV hypertrophy was detected: LV weight/body weight (LVW/BW) ratio (+12.3+/-3%, p < 0.05) and mRNA expression of atrial natriuretic peptide (ANP) in WT mice (+120+/-25%, p < 0.05) after 3 days were increased. In contrast, NE did not induce elevation of LVW/BW ratio and ANP expression in IL-6(-/-) mice. Replacement with recombinant IL-6 restored the hypertrophy-inducing effect of NE in IL-6(-/-) mice. As to the extracellular matrix (ECM) proteins, NE increased collagen type I and III expression only in WT mice and not in IL-6(-/-) mice. The addition of recombinant IL-6 elevated the expression of the ECM proteins to the WT level. CONCLUSION: IL-6 is a major player in the development of NE-induced LV hypertrophy in mice.

Cord blood cell therapy alters LV remodeling and cytokine expression but does not improve heart function after myocardial infarction in rats.

OBJECTIVE: In this study the ability of unrestricted somatic stem cells (USSC) and mononuclear cord blood cells (MN-CBC) was tested to improve heart function and left ventricular (LV) remodeling after myocardial infarction (MI). METHODS: The cells were delivered by i.v. or intramyocardial injections in rat models of MI by permanent coronary artery occlusion and by ischemia/reperfusion (I/R) injury. Heart function and remodeling was followed by recurrent echocardiography over 8 or 12 weeks after which catheterization was performed. RESULTS: Although injected labeled cells could be observed within the myocardium for up to 6 d, there was no sign of cardiac regeneration 8 or 12 weeks after MI. However, the mRNA expression of components of the extracellular matrix was attenuated in the infarct scar 12 weeks after MI and cell injection. Additionally, the expression of interleukin (IL)-6 but not of IL-1 beta increased at the site of injury and the adjacent border-zone 12 weeks after I/R and USSC-injection. However, these effects did not translate into improved heart function or attenuated LV dilatation. CONCLUSION: These data indicate that cord blood cell implantation after MI acts through paracrine mechanisms to modify remodeling rather than myocyte regeneration. The role of myofibroblasts and the optimal conditions of cell application need to be determined to translate these mechanisms into functional improvement.

Tissue inhibitor of matrix metalloproteinase-1 in norepinephrine-induced remodeling of the mouse heart.

BACKGROUND: Matrix metalloproteinases (MMPs) play an important role in myocardial remodeling. Their activity is regulated by the tissue inhibitors of metalloproteinases (TIMPs). The present study analyzed the contribution of changes in functional and molecular parameters to early cardiac remodeling in mice hearts. The role that TIMPs might play in this process was specially acknowledged. METHODS: The remodeling was induced by norepinephrine (NE) given sc in balb/c mice. Varying concentrations, time and the addition of a neutralizing TIMP-1 antibody were evaluated. RESULTS: High dose NE led to insufficiency of the left ventricle (LV) as evidenced by reduced NE-induced elevation of LV systolic pressure, contractility and relaxation. Further, signs of lung congestion were seen. NE induced a concentration-dependent increase of LV weight/body weight (LVW/BW) ratio and elevated mRNA expression of atrial natriuretic peptide (ANP). This was accompanied by induction of collagen type I and III, as well as TIMP-1 expression. CONCLUSIONS: The NE-induced increase of TIMP-1 expression may induce the elevation of the antihypertrophic cardiac factor ANP since NE-induced increase of ANP expression was abolished by neutralizing TIMP-1 antibody. Thus, TIMP-1 may mediate ANP-induced attenuation of NE-induced hypertrophy in the mouse heart.

Time course of hypoxia-induced lung injury in rats.

We investigated the effects of normobaric hypoxia on rat lungs and hypothesized that the hypoxic exposure would induce lung injury with pulmonary edema and inflammation ensued by development of fibrosis. Rats were exposed to 10% O(2) in nitrogen over 6-168h. We analyzed cardiovascular function and pulmonary changes, lung histology and mRNA expression of extracellular matrix (ECM) molecules in the lung. Significant hemodynamic changes occurred after 168h of hypoxic exposure. Moderate pulmonary edema appeared after 8h and peaked after 16h of hypoxia. It was accompanied by inflammation, fibrosis and vascular hypertrophy. mRNA expression of transforming growth factor-beta2 and -beta3 was up-regulated in lung tissue after 8h of hypoxia. After 8-16h, mRNA expression of collagen types I and III and of other ECM molecules was significantly elevated and increased further with longer exposure to hypoxia. The time course of hypoxia-induced pulmonary injury resembled that previously observed after continuous norepinephrine infusion in rats.

Effects of salt loading and various therapies on cardiac hypertrophy and fibrosis in young spontaneously hypertensive rats.

We investigated the effects of salt loading on blood pressure, cardiac hypertrophy and fibrosis as well as on the effectiveness of various antihypertensive therapies in young spontaneously hypertensive rats (SHR). Twenty-five male SHR were salt-stimulated by drinking 1% NaCl from 3 to 6 months of age. Eighteen of them were treated for the last 2 weeks of salt loading with either the angiotensin-converting enzyme inhibitor captopril, the beta-adrenergic receptor blocker propranolol or the calcium-channel antagonist verapamil. Age-matched male Wistar-Kyoto (WKY) rats and SHR drinking only water served as controls. At the age of 6 months, SHR had significantly elevated blood pressure that was unchanged by salt loading. Relative heart weight was increased in SHR without (3.3) and even more so with salt intake (3.6 vs. 2.4 in WKY). Left ventricular (LV) hypertrophy was accompanied by a 17-fold increase in the expression of mRNA for atrial natriuretic factor (ANF) both in untreated and salt-loaded SHR compared to WKY (p<0.001). Collagen I and III mRNA increased 1.7-1.8-fold in SHR without and with additional salt intake (p<0.01). None of the therapies significantly reduced blood pressure or hypertrophy. Although captopril had no antihypertensive effect, it reduced ANF, collagen I and III mRNA in LV to control level. Less pronounced effects were achieved with verapamil. These findings emphasize the cardioprotective role of captopril which may not be fully expressed in the presence of elevated salt intake.

Heart function and molecular biological parameters are comparable in young adult and aged rats after chronic myocardial infarction.

OBJECTIVE: To test the hypothesis that IL-1beta and IL-6 play a pivotal role after myocardial infarction (MI) particularly in aged rats. METHODS: Chronic MI was induced in young adult (3.5 months) and aged (18 months) female Sprague-Dawley rats by ligation of the left coronary artery. Sham-operated animals of corresponding age served as controls. Heart function was measured by catheterization 4 weeks after MI. The expression of IL-1beta, IL-6, TGF-beta-isoforms, ANF, and components of the extracellular matrix (pro-collagen I and III, colligin, MMP-2 and TIMP2) was measured by ribonuclease protection assay. RESULTS: Aged control rats differed from young adult rats in that LV-developed pressure (LVDP) was higher (161 vs. 147 mmHg, p<0.05) in response to the elevated total peripheral resistance (0.71 vs. 0.47 mmHg ml min/kg, p<0.05). Contractility was reduced in aged controls as indicated by decreased LV dP/dt (8.106 vs. 10.606 mmHg/s, p<0.05). LV function was severely depressed in both MI groups (reduction in LVDP by about 35% and LV dP/dt by about 30%, increase in LVEDP to 24 mmHg) while RVP and RV dP/dt markedly increased by about 100%. This was not different between both MI groups. ANF expression as a marker of hypertrophy was induced in both MI groups, but less pronounced in the LV of aged rats. Also, the mRNA expression pattern was qualitatively comparable, but showed gradual differences. CONCLUSION: These results indicate that aged rats compensate well for hemodynamic overload induced by MI. Also, the mechanisms of myocardial post-MI remodeling are comparable in young adult and aged rats.

Hematopoietic stem cells do not repair the infarcted mouse heart.

OBJECTIVE: Recent reports suggest that hematopoietic stem cells (HSC) can transdifferentiate into cardiomyoctes and contribute to myocardial regeneration after injury. This concept has recently been challenged by studies in which bone-marrow (BM)-derived cells do not acquire a cardiac phenotype after direct injection into ischemic myocardium. METHODS: In this study, we analyzed the effect of increased circulating adult BM cells by stimulation with stem cell factor (SCF; 200 microg/kg/d for 7 days) and granulocyte-colony stimulating factor (G-CSF, 50 microg/kg/d for 7 days) or by peripheral delivery of isolated adult BM cells on morphological and hemodynamic parameters of mouse hearts 6 weeks after induction of chronic myocardial infarction (MI). All animals were splenectomized to prevent sequestration of BM cells 2 weeks prior to the induction of MI. Cytokine treatment was initiated either 3 days prior to or 6 h after MI. Isolated, either whole or by magnetic beads lineage-depleted BM cells were injected via a tail vein 6 h after MI. RESULTS: Left and right ventricular (LV and RV) function revealed no improvement in any treatment group when compared to untreated MI animals at baseline resting conditions as well as after stimulation with norepinephrine (NE; 1, 5, 10, 25, 50, and 100 ng bolus i.v. in 10 microl each) as measured by catherization with ultraminiature 1.4 F tip pressure transducers 6 weeks after MI. Moreover, there was no sign of myocardial regeneration in histological or gene expression analyses. CONCLUSION: Mobilization or i.v. injection of BM cells do not have a measurable effect on cardiac regeneration.

Mechanism of cell death of rat cardiac fibroblasts induced by serum depletion.

Serum starvation has recently been shown to cause cell death of cardiac fibroblasts and increased synthesis of extracellular matrix proteins in the surviving cells. In the present study, events occurring in the dying cells were investigated. Cultured adult rat cardiac fibroblasts were exposed to serum-free medium. Cell number was measured using a Coulter Counter Channelyzer. The activity of the extracellular signal-regulated or mitogen-activated protein kinases (ERK1/2, p42/p44(MAPK)), the p38 kinase (p38(MAPK)), the c-Jun N-terminal kinases (p46/p54(JNK)), and Akt kinase was assessed by Western blotting and phospho-specific antibodies. Caspase 7-cleavage was investigated by Western blotting and specific antibodies. Caspase 3 activity was measured by detection of its cleaved substrate. The appearance of necrosis was studied by inclusion of trypan blue. Apoptosis was assessed by DNA ladder formation. The mRNA expression of Bax and Bcl-2 was investigated by quantitative real-time PCR. Serum withdrawal led to the death of 26% of cultured isolated cardiac fibroblasts during the first 5 h. The activity of the p42/ p44(MAPK) as well as of Akt kinase was partially reduced. For p46/p54(JNK) and p38(MAPK), elevated phosphorylation was measured. Inhibition of p46/p54(JNK) and p38(MAPK) activity by SB202190 did not affect the decrease in cell number. Cleavage of caspase 7 was detected after 90 min. However, no activation of caspase 3 was measured. DNA fragmentation was not found after serum depletion. Trypan blue staining, however, was observed in 16% of the cells after 5 h. The mRNA levels of both Bax and Bcl-2 were increased after 30 min. These results indicate the appearance of necrosis during serum starvation in cardiac fibroblasts. However, some processes typical of apoptosis were also detected.