A randomised preclinical trial of adrenaline use during cardiac arrest in mice.

Background: Adrenaline is routinely administered during cardiac arrest resuscitation. Using a novel murine model of cardiac arrest, this study evaluates the effects of adrenaline use on survival and end-organ injury. Methods: A total of 58 mice, including cardiac arrest (CA) and sham (SHAM) groups received intravenous potassium chloride either as a bolus (CA) or slow infusion (SHAM), inducing ECG-confirmed asystole (in CA only) for 4-minutes prior to intravenous adrenaline (+ADR;250 ul,32 ug/ml) or saline (-ADR;250 ul) and manual chest compressions (300 BPM) for 4-minutes. Mice with return of spontaneous circulation (ROSC) were assessed at 24- or 72-h timepoints. Results: Among animals that underwent CA, rates of ROSC (n = 21 (95 %) vs n = 14 (82 %), P = 0.18) and survival to the planned endpoint (n = 11 (50 %) vs n = 12 (71 %), P = 0.19) were similar when comparing those treated with (CA+ADR) and without (CA-ADR) adrenaline. However, in CA animals that initially achieved ROSC, subsequent mortality was approximately 3-fold greater with adrenaline treatment (48 % vs 14 %, P = 0.042). Among SHAM animals, adrenaline use had no impact on survival rates or other endpoints. Greater myocardial injury occurred in CA+ADR vs CA-ADR, with increased Hs-Troponin levels measured at 24- (26.0 ± 0.9 vs 9.4 ± 5.3 ng/mL, P = 0.015) and 72-h (20.9 ± 8.3 vs 5.0 ± 2.4 ng/mL, P = 0.012), associated with increased expression of pro-inflammatory and fibrotic genes within cardiac and renal tissue. Conclusion: Adrenaline did not improve ROSC or overall survival but following successful ROSC, its use resulted in 3-fold greater mortality rates. Adrenaline was also associated with increased myocardial injury, end-organ inflammation, and fibrosis. These findings underscore the need for further preclinical evaluation of alternate pharmacologic adjuncts for cardiopulmonary resuscitation that improve survival and limit end-organ injury.

Endothelial-specific overexpression of cationic amino acid transporter-1 prevents loss of kidney function in heart failure.

Heart failure (HF) is associated with impaired L-arginine transport. In the present study, we tested the hypothesis that augmented L-arginine transport prevents the loss of kidney function in HF. Renal function was assessed in wildtype mice (WT), transgenic mice with HF (dilated cardiomyopathy, DCM) and double transgenic mice (double transgenic mice with DCM and CAT-1 overexpression, HFCAT-1) with HF and endothelial-specific overexpression of the predominant L-arginine transporter, cationic amino acid transporter-1 (CAT-1) (n=4-8/group). Cardiac function was assessed via echocardiography and left ventricular catheterisation. Renal function was assessed via quantification of albuminuria and creatinine clearance. Plasma nitrate and nitrite levels together with renal fibrosis and inflammatory markers were also quantified at study end. Albumin/creatinine ratio was two-fold greater in DCM mice than in WT mice (P=0.002), and tubulointerstitial and glomerular fibrosis were approximately eight- and three-fold greater, respectively, in DCM mice than in WT mice (P≤0.02). Critically, urinary albumin/creatinine ratio and tubulointerstitial and glomerular fibrosis were less in HFCAT-1 mice than in DCM mice (P<0.05). Renal CAT-1 expression and plasma nitrate and nitrite levels were less in DCM mice compared with WT (P≤0.03) but was greater in HFCAT-1 mice than in DCM mice (P≤0.009). Renal expression of IL-10 was less in DCM mice compared with WT (P<0.001) but was greater in HFCAT-1 mice compared with DCM mice (P=0.02). Our data provide direct evidence that augmented L-arginine transport prevents renal fibrosis, inflammation and loss of kidney function in HF.

CXCR4 Antagonism Reduces Cardiac Fibrosis and Improves Cardiac Performance in Dilated Cardiomyopathy.

Background: Myocardial fibrosis is a key pathologic finding in the failing heart and is implicated as a cause of increased ventricular stiffness and susceptibility to ventricular arrhythmia. Neurohormonal mediators such as aldosterone and angiotensin II are known to cause fibrosis in experimental models, however, clinical evidence for the reversal of fibrosis with relevant antagonists is limited. Recent studies suggest that inflammatory mediators may contribute to fibrosis. In dilated cardiomyopathy the mechanism for myocardial fibrosis is unclear and its implications on systolic function are not known. Methods and Results: We studied the effect of a highly selective antagonist of SDF-1/CXCR4 signaling, AMD3100, on the development of cardiac fibrosis and cardiac function in mice with dilated cardiomyopathy due to cardiac-specific transgenic overexpression of the stress-kinase, Mst1. AMD3100 significantly attenuated the progression of myocardial fibrosis and this was accompanied by significant improvements in diastolic and systolic performance as evaluated in isolated Langendorff perfused hearts. AMD3100 reduced BNP mRNA expression but did not alter the expression of Ca2+ handling genes. CXCR4 antagonism also reduced the abundance of splenic CD4+ T cells. Conclusion: This study demonstrates that CXCR4 pathway contributes to pathogenesis of cardiac fibrosis in dilated cardiomyopathy, and it represents a new potential therapeutic target in heart failure. The data also demonstrate that anti-fibrotic strategies can improve systolic performance.

Serelaxin attenuates renal inflammation and fibrosis in a mouse model of dilated cardiomyopathy.

NEW FINDINGS: What is the central question of this study? The aim was to determine the renoprotective effects of serelaxin in the setting of chronic heart failure. What are the main findings and its importance? Our data indicate that serelaxin can reduce renal fibrosis and inflammation in experimental heart failure. Currently, there are no effective treatments to rescue renal function in heart failure patients, and our data suggest that serelaxin might have the potential to reduce renal fibrosis and inflammation in heart failure. ABSTRACT: Serelaxin has been demonstrated to attenuate renal fibrosis and inflammation in cardiorenal disease. In the present study, we tested the hypothesis that serelaxin can prevent the decline in renal function in dilated cardiomyopathy (DCM) by targeting renal fibrosis and inflammation. Male transgenic mice with DCM (n = 16) and their wild-type littermates (WT; n = 20) were administered either vehicle or serelaxin (500 µg kg-1  day-1 ; subcutaneous minipumps; 8 weeks). Cardiac function was assessed via echocardiography before and during the eighth week of serelaxin treatment. Renal function and inflammation as well as cardiac and renal fibrosis were assessed at the end of the study. Serelaxin had minimal effect on cardiac function (P ≥ 0.99). Tubulointerstitial and glomerular fibrosis were ∼3-fold greater in vehicle-treated DCM mice compared with vehicle-treated WT mice (P ≤ 0.001). Renal mRNA expression of Tnfα and Il1α were ∼4- and ∼3-fold greater, respectively, in vehicle-treated DCM mice compared with vehicle-treated WT mice (P ≤ 0.05). Tubulointerstitial and glomerular fibrosis were 46 and 45% less, respectively, in serelaxin-treated DCM mice than in vehicle-treated DCM mice (P ≤ 0.01). Renal cortical mRNA expression of Tnfα and Il1α were 56 and 58% less, respectively, in the former group compared with the latter (P ≤ 0.05). The urinary albumin:creatinine ratio was ∼3-fold greater in vehicle-treated DCM mice compared with vehicle-treated WT mice (P = 0.02). The urinary albumin:creatinine ratio was not significantly different between vehicle-treated DCM mice and serelaxin-treated DCM mice (P = 0.38). These data suggest that serelaxin can attenuate renal fibrosis and inflammation and has the potential to exert renoprotective effects in DCM.

Age-Related Differential Structural and Transcriptomic Responses in the Hypertensive Heart.

While aging is a critical risk factor for heart failure, it remains uncertain whether the aging heart responds differentially to a hypertensive stimuli. Here we investigated phenotypic and transcriptomic differences between the young and aging heart using a mineralocorticoid-excess model of hypertension. Ten-week ("young") and 36-week ("aging") mice underwent a unilateral uninephrectomy with deoxycorticosterone acetate (DOCA) pellet implantation (n = 6-8/group) and were followed for 6 weeks. Cardiac structure and function, blood pressure (BP) and the cardiac transcriptome were subsequently examined. Young and aging DOCA mice had high BP, increased cardiac mass, cardiac hypertrophy, and fibrosis. Left ventricular end-diastolic pressure increased in aging DOCA-treated mice in contrast to young DOCA mice. Interstitial and perivascular fibrosis occurred in response to DOCA, but perivascular fibrosis was greater in aging mice. Transcriptomic analysis showed that young mice had features of higher oxidative stress, likely due to activation of the respiratory electron transport chain. In contrast, aging mice showed up-regulation of collagen formation in association with activation of innate immunity together with markers of inflammation including cytokine and platelet signaling. In comparison to younger mice, aging mice demonstrated different phenotypic and molecular responses to hypertensive stress. These findings have potential implications for the pathogenesis of age-related forms of cardiovascular disease, particularly heart failure.

N-Acetylcysteine Attenuates the Development of Renal Fibrosis in Transgenic Mice with Dilated Cardiomyopathy.

Mechanisms underlying the renal pathology in cardiorenal syndrome (CRS) type 2 remain elusive. We hypothesised that renal glutathione deficiency is central to the development of CRS type 2. Glutathione precursor, N-acetylcysteine (NAC;40 mg/kg/day; 8 weeks) or saline were administered to transgenic mice with dilated cardiomyopathy (DCM) and wild-type (WT) controls. Cardiac structure, function and glutathione levels were assessed at the end of this protocol. Renal fibrosis, glutathione content, expression of inflammatory and fibrotic markers, and function were also evaluated. In both genotypes, NAC had minimal effect on cardiac glutathione, structure and function (P ≥ 0.20). In NAC treated DCM mice, loss of glomerular filtration rate (GFR), tubulointerstitial and glomerular fibrosis and renal oxidised glutathione levels were attenuated by 38%, 99%, 70% and 52% respectively, compared to saline treated DCM mice (P ≤ 0.01). Renal expression of PAI-1 was greater in saline treated DCM mice than in WT mice (P < 0.05). Renal PAI-1 expression was less in NAC treated DCM mice than in vehicle treated DCM mice (P = 0.03). Renal IL-10 expression was greater in the former cohort compared to the latter (P < 0.01). These data indicate that normalisation of renal oxidized glutathione levels attenuates PAI-1 expression and renal inflammation preventing loss of GFR in experimental DCM.

High-Fiber Diet and Acetate Supplementation Change the Gut Microbiota and Prevent the Development of Hypertension and Heart Failure in Hypertensive Mice.

BACKGROUND: Dietary intake of fruit and vegetables is associated with lower incidence of hypertension, but the mechanisms involved have not been elucidated. Here, we evaluated the effect of a high-fiber diet and supplementation with the short-chain fatty acid acetate on the gut microbiota and the prevention of cardiovascular disease. METHODS: Gut microbiome, cardiorenal structure/function, and blood pressure were examined in sham and mineralocorticoid excess-treated mice with a control diet, high-fiber diet, or acetate supplementation. We also determined the renal and cardiac transcriptome of mice treated with the different diets. RESULTS: We found that high consumption of fiber modified the gut microbiota populations and increased the abundance of acetate-producing bacteria independently of mineralocorticoid excess. Both fiber and acetate decreased gut dysbiosis, measured by the ratio of Firmicutes to Bacteroidetes, and increased the prevalence of Bacteroides acidifaciens. Compared with mineralocorticoid-excess mice fed a control diet, both high-fiber diet and acetate supplementation significantly reduced systolic and diastolic blood pressures, cardiac fibrosis, and left ventricular hypertrophy. Acetate had similar effects and markedly reduced renal fibrosis. Transcriptome analyses showed that the protective effects of high fiber and acetate were accompanied by the downregulation of cardiac and renal Egr1, a master cardiovascular regulator involved in cardiac hypertrophy, cardiorenal fibrosis, and inflammation. We also observed the upregulation of a network of genes involved in circadian rhythm in both tissues and downregulation of the renin-angiotensin system in the kidney and mitogen-activated protein kinase signaling in the heart. CONCLUSIONS: A diet high in fiber led to changes in the gut microbiota that played a protective role in the development of cardiovascular disease. The favorable effects of fiber may be explained by the generation and distribution of one of the main metabolites of the gut microbiota, the short-chain fatty acid acetate. Acetate effected several molecular changes associated with improved cardiovascular health and function.

N-acetylcysteine attenuates the development of cardiac fibrosis and remodeling in a mouse model of heart failure.

Oxidative stress plays a central role in the pathogenesis of heart failure. We aimed to determine whether the antioxidantN-acetylcysteine can attenuate cardiac fibrosis and remodeling in a mouse model of heart failure. Minipumps were implanted subcutaneously in wild-type mice (n = 20) and mice with cardiomyopathy secondary to cardiac specific overexpression of mammalian sterile 20-like kinase 1 (MST-1;n = 18) to administerN-acetylcysteine (40 mg/kg per day) or saline for a period of 8 weeks. At the end of this period, cardiac remodeling and function was assessed via echocardiography. Fibrosis, oxidative stress, and expression of collagen types I andIIIwere quantified in heart tissues. Cardiac perivascular and interstitial fibrosis were greater by 114% and 209%, respectively, inMST-1 compared to wild type (P ≤ 0.001). InMST-1 mice administeredN-acetylcysteine, perivascular and interstitial fibrosis were 40% and 57% less, respectively, compared to those treated with saline (P ≤ 0. 03). Cardiac oxidative stress was 119% greater inMST-1 than in wild type (P < 0.001) andN-acetylcysteine attenuated oxidative stress inMST-1 by 42% (P = 0.005). These data indicate thatN-acetylcysteine can blunt cardiac fibrosis and related remodeling in the setting of heart failure potentially by reducing oxidative stress. This study provides the basis to investigate the role ofN-acetylcysteine in chronic heart failure.

CXCR4 Antagonism Attenuates the Development of Diabetic Cardiac Fibrosis.

Heart failure (HF) is an increasingly recognized complication of diabetes. Cardiac fibrosis is an important causative mechanism of HF associated with diabetes. Recent data indicate that inflammation may be particularly important in the pathogenesis of cardiovascular fibrosis. We sought to determine the mechanism by which cardiac fibrosis develops and to specifically investigate the role of the CXCR4 axis in this process. Animals with type I diabetes (streptozotocin treated mice) or type II diabetes (Israeli Sand-rats) and controls were randomized to treatment with a CXCR4 antagonist, candesartan or vehicle control. Additional groups of mice also underwent bone marrow transplantation (GFP+ donor marrow) to investigate the potential role of bone marrow derived cell mobilization in the pathogenesis of cardiac fibrosis. Both type I and II models of diabetes were accompanied by the development of significant cardiac fibrosis. CXCR4 antagonism markedly reduced cardiac fibrosis in both models of diabetes, similar in magnitude to that seen with candesartan. In contrast to candesartan, the anti-fibrotic actions of CXCR4 antagonism occurred in a blood pressure independent manner. Whilst the induction of diabetes did not increase the overall myocardial burden of GFP+ cells, it was accompanied by an increase in GFP+ cells expressing the fibroblast marker alpha-smooth muscle actin and this was attenuated by CXCR4 antagonism. CXCR4 antagonism was also accompanied by increased levels of circulating regulatory T cells. Taken together the current data indicate that pharmacological inhibition of CXCR4 significantly reduces diabetes induced cardiac fibrosis, providing a potentially important therapeutic approach.

CXCR4 antagonism attenuates the cardiorenal consequences of mineralocorticoid excess.

BACKGROUND: Extensive evidence implicates aldosterone excess in the development and progression of cardiovascular disease states including hypertension, metabolic syndrome, cardiac hypertrophy, heart failure, and cardiorenal fibrosis. Recent studies show that activation of inflammatory cascade may play a specific role in the sequelae of mineralocorticoid activation, although the linking mechanism remains unclear. We tested the possibility that secondary stimulation of the stromal-derived factor 1/CXC chemokine receptor 4 (SDF-1/CXCR4) pathway plays a contributory role. METHODS AND RESULTS: We investigated the effect of the highly selective CXCR4 antagonist AMD3465 (6 mg/kg per day for 6 weeks through minipump) in dexoycorticosterone acetate (DOCA)-treated, uninephrectomized mice. CXCR4 antagonism significantly attenuated the induction of cardiac fibrosis, renal fibrosis, hypertension, and left ventricular hypertrophy by DOCA. Mineralocorticoid excess also stimulated the accumulation of T-lymphocytes in the heart and kidney and this was significantly blunted by CXCR4 inhibition. CONCLUSIONS: Taken together, these data strongly implicate the SDF-1/CXCR4 axis in the pathogenesis of mineralocorticoid excess induced hypertension, inflammation, and cardiorenal fibrosis. This insight provides a new potential therapeutic approach for the treatment of specific aspects of mineralocorticoid mediated cardiovascular disease.

Bone marrow-derived cells contribute to fibrosis in the chronically failing heart.

Cardiac fibrosis contributes significantly to the phenotype of the chronically failing heart. It is not clear whether in this setting the fibrosis is contributed by native cardiac fibroblasts or alternatively by recruitment of cells arising from the bone marrow. We aimed to determine the contribution of bone marrow-derived cells to cardiac fibrosis in the failing heart and to investigate potentially contributing cytokines. Bone marrow-derived fibrocyte recruitment to the failing heart was studied in a transgenic (Mst1 mice) model of dilated cardiomyopathy. In conjunction, we examined the role of stromal-derived factor-1 (SDF-1), a key chemoattractant, by assessing myocardial expression and secretion by cardiomyocytes and in clinical samples. Bone marrow-derived cells were recruited in significantly greater numbers in Mst1 versus control mice (P < 0.001), contributing 17 +/- 4% of the total fibroblast load in heart failure. Patients with heart failure had higher plasma levels of SDF-1 than healthy control subjects (P < 0.01). We found that cardiomyocytes constitutively secrete SDF-1, which is significantly up-regulated by angiotensin II. SDF-1 was shown to increases cardiac fibroblast migration by 59% (P < 0.05). Taken together, our data suggest that recruitment of bone marrow-derived cells under the influence of factors, including SDF-1, may play an important role in the pathogenesis of cardiac fibrosis in heart failure.

Mice deficient for the chromosome 21 ortholog Itsn1 exhibit vesicle-trafficking abnormalities.

Enlarged early endosomes in the neurons of young Down syndrome (DS) and pre-Alzheimer's disease (AD) brains suggest that a disturbance in endocytosis is one of the earliest hallmarks of AD pathogenesis in both conditions. We identified a chromosome 21 gene, Intersectin-1 (ITSN1) that is up-regulated in DS brains and has a putative function in endocytosis and vesicle trafficking. To elucidate the function of ITSN1 and assess its contribution to endocytic defects associated with DS and AD, we generated Itsn1 null mice. In knockout mice we found alterations in a number of parameters associated with endocytic and vesicle trafficking events. We found a reduced number of exocytosis events in chromaffin cells and a slowing of endocytosis in neurons. Endosome size was increased in neurons and NGF levels were reduced in the septal region of the brain. Our data is the first indication that Itsn1 has a role in endocytosis in an in vivo mammalian model, and that a disruption in Itsn1 expression causes a disturbance in vesicle trafficking and endocytic function in the brain. These results imply a role for ITSN1 in the early endocytic anomalies reported in DS brains which may have ramifications for the onset of AD.

TGF-betaRII rescues development of small intestinal epithelial cells in Elf3-deficient mice.

BACKGROUND & AIMS: ELF3, a member of the ETS transcription factor family, has been shown to transactivate the transforming growth factor beta type II receptor (TGF-betaRII) promoter. Previously we showed that Elf3-null mice have a defect in the small intestine caused by a failure of small intestinal epithelial cells to differentiate and that these cells produced significantly lower levels of Tgf-betaRII. To prove that the defect observed in Elf3-null mice resulted from the lack of Elf3-dependent activation of Tgf-betaRII expression, we performed a genetic rescue. METHODS: We generated transgenic mice that express human TGF-betaRII specifically in the intestinal epithelium under the control of the mouse A33 antigen promoter. Mice expressing the A33-TGF-betaRII transgene were mated with Elf3(+/-) mice, and double heterozygous offspring harboring both the transgene and one mutant Elf3 allele were intercrossed. RESULTS: The resultant A33-TGF-betaRII transgenic Elf3(-/-) pups displayed normal small intestinal morphology, while the characteristic abnormality was retained in all Elf3(-/-) mice that did not express the transgene. This phenotypic rescue shows for the first time in vivo that a single gene, Elf3, is the critical upstream regulator of Tgf-betaRII in mouse small intestinal epithelium. CONCLUSIONS: This has important implications for our understanding of tissue-specific gene regulation and further strengthens the potential clinical connection between ELF3 and colorectal cancer involving transforming growth factor beta insensitivity.

Degeneration and apoptosis of endometrial cells in the bitch.

The relationships between changes in plasma progesterone concentrations, degeneration of the luminal epithelium, the occurrence of apoptosis of endometrial cells and endometrial leucocyte populations in the bitch were determined. Mature bitches (n = 15) were euthanized and necropsied when in diestrus (Days 7-75, n = 12) or in anestrus (Days 10, 32 and 53). Degeneration of the luminal epithelium was observed in bitches in late diestrus (Days 38-75, n = 5) when plasma progesterone concentrations were decreasing and in anestrus (Days 10 and 32, n = 2) when plasma progesterone concentrations were < 0.5 ng/mL. Endometrial leucocyte populations increased after degeneration of the luminal epithelium (around Day 42 of diestrus). Apoptosis was mainly observed in the basal glandular epithelial cells and endothelial cells of blood capillaries in all except anestrous bitches. Very few apoptotic cells were found in the superficial glandular epithelial cells and stromal cells. Higher apoptotic indices were detected in the basal glandular epithelium on Days 12-42 of diestrus than at other stages. Therefore, apoptosis of glandular basal epithelial cells occurred mainly in early diestrus, degeneration of cells of the luminal epithelium occurred from mid-diestrus to early anestrus, and the increase in leucocyte numbers may have been a consequence and not a cause of luminal epithelial degeneration.

WNK1 activates SGK1 to regulate the epithelial sodium channel.

WNK (with no lysine [K]) kinases are serine-threonine protein kinases with an atypical placement of the catalytic lysine. Intronic deletions increase the expression of WNK1 in humans and cause pseudohypoaldosteronism type II, a form of hypertension. WNKs have been linked to ion carriers, but the underlying regulatory mechanisms are unknown. Here, we report a mechanism for the control of ion permeability by WNK1. We show that WNK1 activates the serum- and glucocorticoid-inducible protein kinase SGK1, leading to activation of the epithelial sodium channel. Increased channel activity induced by WNK1 depends on SGK1 and the E3 ubiquitin ligase Nedd4-2. This finding provides compelling evidence that this molecular mechanism contributes to the pathogenesis of hypertension in pseudohypoaldosteronism type II caused by WNK1 and, possibly, in other forms of hypertension.

Dietary potassium restriction stimulates endocytosis of ROMK channel in rat cortical collecting duct.

ROMK potassium channels are present in the cortical collecting ducts (CCDs) of the kidney and serve as the exit pathways for K+ secretion in this nephron segment. Dietary K+ restriction reduces the abundance of ROMK in the kidney. We have previously shown that ROMK undergoes endocytosis via clathrin-coated vesicles in Xenopus laevis oocytes and in cultured cells. Here, we examined the effect of dietary K+ restriction on endocytosis of ROMK in CCDs using double-labeling immunofluorescent staining and confocal microscopic imaging in whole kidney sections as well as in individually isolated tubules. We found that ROMK abundance in kidney cortex and CCDs was reduced in rats fed a K+-restricted diet compared with rats fed the control K+ diet. In the control animals, ROMK staining was preferentially localized to the apical membrane of CCDs. Compared with control tubules, ROMK staining in CCDs was markedly shifted toward intracellular locations in animals fed a K+-deficient diet for 48 h. Some of the intracellular distribution of ROMK colocalized with an early endosomal marker, early endosomal antigen-1 or with a late endosomal/lysosomal marker, lysosomal membrane glycoprotein-120. These results suggest that K+ restriction reduces the abundance of ROMK in CCDs by increasing endocytosis and degradation of the channel protein. This decrease in the abundance of ROMK is likely important for maintaining K+ homeostasis during K+ deficiency.

Apoptosis of endometrial cells in the bitch.

The relationship between apoptosis of endometrial cells in the bitch, and the occurrence of degeneration of the endometrial luminal epithelium and regression of the endometrial glandular epithelium was determined. Mature bitches (n = 12) were ovariectomized and treated with hormones to simulate uterine changes that occur during the oestrous cycle. All bitches were treated with oestradiol benzoate (0.6-4.8 microg x kg(-1), i. m., twice per day) for 11-12 days then with progestagen (megestrol acetate, 2 mg x kg(-1), p. o., once per day) for 35-37 days. Bitches were treated daily for a further 3 weeks with megestrol acetate at dose rates of 0.5 mg x kg(-1) (decreased-dose group (n = 3)), 2 mg x kg(-1) (standard-dose group (n = 3)), or 3 (1 wk), 4 (1 wk) and 5 (1 wk) mg x kg(-1) (increased-dose group (n = 3)), or received no treatment (withdrawal-dose group (n = 3)). These bitches were necropsied at the end of the treatment period. A further 10, ovary-intact, bitches were necropsied when in oestrus (n = 1), dioestrus (n = 5), and at 3 weeks (n = 1) and 9 weeks (n = 3) of anoestrus. Degeneration of the luminal epithelium was observed in all bitches except those in oestrus, at 9 weeks of anoestrus and one in dioestrus. Apoptosis was observed in the glandular epithelial cells, stromal cells and endothelial cells of blood capillaries in all bitches except those at oestrous and at 9 weeks of anoestrous. High average apoptotic indices were detected in the basal glandular epithelium of the dioestrous, decreased-dose, standard-dose and increased-dose groups, whereas low apoptotic indices were detected at 3 weeks of anoestrous and in the withdrawal-dose groups. These results indicate that degeneration of cells of the glandular epithelium, but not of the luminal epithelium, was due to apoptosis of these cells.