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Biomedical subjects

Robert E Leggett

Publications and source records attributed to Robert E Leggett.

12 recordsLinked to original sources

Effect of bilateral in vivo ischemia/reperfusion on the activities of superoxide dismutase and catalase: response to a standardized grape suspension.

PURPOSE: Ischemia/reperfusion (I/R) is a major etiological factor in the bladder dysfunctions observed in men with lower tract obstruction, women with postmenopausal incontinence and with aging. A standardized grape suspension protects the rabbit urinary bladder from both the contractile dysfunctions and the morphologic changes mediated by I/R. Using a model of in vivo bilateral ischemia/reperfusion, the current study investigated the effect of this grape suspension on the endogenous antioxidant defense systems. MATERIALS AND METHODS: 24 NZW rabbits were separated into 6 groups of 4. Groups 1-3 were treated by gavage with aqueous grape suspensions; groups 4-6 received sugar-water vehicle. Groups 3 and 6 were controls. Groups 1 and 4 were subjected to bilateral ischemia for 2 h (I). Groups 2 and 5 underwent bilateral ischemia for 2 h and reperfusion for 1 week (I/R). For all rabbit bladders, the muscle and mucosa were separated by blunt dissection and analyzed separately. The effects of the various treatments on bladder antioxidant systems of cytoplasmic superoxide dismutase (Cu-Zn superoxide dismutase; SOD), and catalase (CAT) were evaluated. RESULTS: The standardized grape suspension up-regulated both SOD and CAT activity of bladder muscle and mucosa in control animals. There were few differences in the grape suspension treated animals after ischemia, and in general the activities decreased following I/R. CONCLUSIONS: Increases of SOD and CAT activity in control animals as a result of grape suspension suggest a greater antioxidant capacity. This increase in the antioxidant defense system may explain the increased protection of grape suspension in the face of ischemia and I/R. However, the activities of both enzyme systems decreased in the smooth muscle subjected to I/R showing that reperfusion damages these systems probably via oxidation damage to the enzymes themselves.

Animals↗

The effects of cyclical oestrogen on bladder and urethral structure and function.

OBJECTIVE: To determine the effects of cycling oestrogen in rabbits, as oestrogen is essential for physiological maintenance and integrity of the female urogenital tract. MATERIALS AND METHODS: Changes in circulating oestrogen have marked effects on the bladder of experimental animals, with ovariectomy (Ovx) inducing smooth muscle (SM) and mucosal atrophy, increasing collagen synthesis and deposition, decreasing contractile function, mucosal and SM blood flow; oestrogen reverses these effects and increases bladder mass and SM density, primarily by stimulating angiogenesis and increasing blood flow. Twenty adult female New Zealand White rabbits were divided into five equal groups; group 1 served as the control group, and groups 2-5 had a bilateral Ovx. Group 2 received no oestradiol and was assessed 2 weeks after Ovx; groups 3-5 received 17-beta oestradiol from a subcutaneous slow-release tablet 2 weeks after Ovx, which remained in place for a subsequent 2-week period. Group 3 was then assessed after the 2 weeks on oestradiol. Groups 4 and 5 then had their oestradiol tablets removed for 2 weeks and group 4 was assessed after this period off oestradiol. Group 5 then received a new oestradiol tablet that was left in place for an additional 2 weeks. RESULTS: Both groups receiving oestrogen (3 and 5) had a statistically significantly greater bladder weight than both the control group and group 2. The volume fraction of SM paralleled the bladder weight, showing that oestrogen increased the volume fraction of SM whereas Ovx and low oestrogen decreased the SM fraction. The cross-sections of the urethra from groups 3 and 5 were significantly wider than those of either the control or group 1, also being consistent with the structural effects of oestrogen. Intra-arterial phenylephrine increased urethral pressure to a similar level in all groups. The urethral pressure response to intra-arterial acetylcholine shifted from contraction in the control to relaxation in the oestrogen-treated groups. Ovx resulted in a lower vascular density, whereas oestrogen resulted in a significant increase in vascular density (angiogenesis). CONCLUSIONS: Cyclical oestrogen had pronounced structural and pharmacological effects. Low oestrogen decreased the volume fraction of SM, increased collagen, and decreased vasculature, whereas oestrogen mediated a marked hypertrophy of the SM components, decreased the collagen component, and stimulated angiogenesis. Cyclical oestrogen also had marked effects on the responses to intra-arterial acetylcholine, shifting the response from contraction to relaxation.

Animals↗

Alteration of contractile and regulatory proteins in estrogen-induced hypertrophy of female rabbit bladder.

OBJECTIVES: Estrogen is essential to mediate physiologic functions in female bladders. Deficiency of estrogen has been speculated to be an etiologic factor for bladder dysfunction in postmenopausal women. Our previous studies have demonstrated that estrogen supplementation in female rabbits induces a "functional hypertrophy" of the urinary bladder smooth muscle. The present study investigated the alterations in the contractile and regulatory proteins in this model. METHODS: Twenty New Zealand white female rabbits were separated into five groups of 4 rabbits each. Group 1 served as the control, groups 2 to 6 underwent ovariectomy (Ovx), and group 2 served as the Ovx without estradiol treatment group. Two weeks after Ovx, groups 3 to 5 were given 17-beta estradiol (1 mg/kg/day) by subcutaneous implant for 1, 3, and 7 days, respectively. The expression of the contractile and regulatory proteins, such as myosin light chain kinase, rho-kinase, and caldesmon, was analyzed by Western blotting. RESULTS: The expression of myosin light chain kinase was enhanced by estradiol supplementation. The expression of rho-kinase-alpha was increased significantly (20-fold) after Ovx, which was downregulated after estrogen supplementation. No significant change was seen in rho-kinase-beta after Ovx or estradiol supplementation. The expression of caldesmon isoforms was enhanced by 1-day estradiol supplementation but decreased to lower levels than those of the control group by 3 and 7 days of estrogen treatment. CONCLUSIONS: The results of the present study have provided more understanding about the role of the contractile and regulatory proteins in detrusor muscle, in both dysfunctional atrophy induced by Ovx and functional hypertrophy induced by estrogen supplementation.

Animals↗

Estrogen induces angiogenesis of the female rabbit bladder.

Postmenopausal bladder dysfunction has been speculated to involve decreased circulating estrogen levels. It is our hypothesis that estrogen induces bladder dysfunctions by modulating blood flow to the bladder, i.e. low estrogen reduces blood flow to the bladder, whereas high estrogen increases blood flow. Our previous studies have demonstrated that estrogen administration in female rabbits induces a 'functional hypertrophy' of the urinary bladder smooth muscle represented by increased smooth muscle mass, which corresponds to increased contractile responses to all forms of stimulation. The present study investigates the effect of estrogen on vasculature density and distribution. Twenty-four female New Zealand white rabbits were separated into six groups of four rabbits each. Group 1 served as controls. Groups 2-6 were ovariectomized. Two weeks after ovariectomy (Ovx), groups 3-6 were given 17-beta estradiol (1 mg/kg per day) by s.c. implant for 1, 3, 7, and 14 days respectively. Blood vessel density and distribution were evaluated by immunohistochemistry and quantitative image analyses. Ovx resulted in significant vascular degeneration and decreased density, whereas estradiol administration mediated a significant angiogenic effect characterized by increased vascular density, and distribution of new vasculature within the smooth muscle bundles of the detrusor. Estradiol-induced vasculogenesis corresponds with our previously demonstrated increase in blood flow to the bladder and increased contractility. The most interesting aspect of these studies is the increased vascularization localized within the muscle bundles rather than between the muscle bundles, which may be important in the link between estrogen and increased incidence of cancers.

Animals↗

Effect of partial outlet obstruction on nitrotyrosine content and distribution within the rabbit bladder.

PURPOSE: Evidence indicates that free radicals are etiological factors in obstructive bladder disease. However, it is not clear which species of reactive oxygen or nitrogen species mediate the damage. The current studies were designed to determine if partial outlet obstruction in rabbits results in the generation of nitrotyrosine (NT). MATERIALS AND METHODS: Sixteen rabbits were separated into four groups of four. The rabbits in groups 1 and 2 underwent sham operation while rabbits in groups 3 and 4 underwent partial outlet obstruction. The rabbits in groups 1 and 3 were evaluated after 1 week of obstruction and the rabbits in groups 2 and 4 were evaluated after 2 weeks of obstruction. A separate group of four controls were evaluated simultaneously with the sham and obstructed rabbits. Four rabbits from each group were evaluated after 1 and 2 weeks of obstruction. Four control rabbits were also evaluated. Isolated strips were evaluated for contractile responses and NT content of the mucosa and muscle were quantitated by Western blot analysis. RESULTS: (1) The mucosa contains both 42 and 62 kD proteins exhibiting a strong nitrotyrosine signal; the muscle presents a signal only at 62 kD. (2) The sham operations had no effect on nitrotyrosine distribution or content. (3) The nitrotyrosine of both mucosal proteins and the muscle protein are increased in the 1 week obstructed bladder; whereas, only the 62 kD signal is increased in the two week obstructed bladder mucosa. (4) The contractile response to FS are reduced to a significantly greater degree than the responses to carbachol, KCl, or ATP. CONCLUSIONS: These studies clearly demonstrated that partial outlet obstruction in rabbits results in significant increases in nitrotyrosine within the bladder and may contribute to the contractile dysfunctions mediated by partial outlet obstruction.

Animals↗

Oral Kohki Tea and its protective effect against in vitro ischemic damage to the bladder.

AIMS: Results of several studies indicate that ischemia/reperfusion (I/R) is an etiological factor in the contractile dysfunctions induced by partial bladder outlet obstruction in animal models. In support of this hypothesis, pretreatment of rabbits with Kohki Tea (Engelhardtia chrysolepis), a Japanese herbal drink very high in antioxidant activity, significantly reduced the contractile dysfunctions induced by partial outlet obstruction. The current study was designed to determine if pretreating rabbits with Kohki Tea could protect the bladder against the contractile damage induced by in vitro ischemia followed by re-oxygenation. METHODS: Forty-eight New Zealand White rabbits were separated into two groups of 24; Group 1 was pretreated by oral gavage for 3 weeks with Kohki Tea and Group 2 received vehicle (water). Each rabbit was anesthetized with pentobarbital. The urinary bladder was rapidly removed and eight longitudinal muscle strips were cut from the bladder body. Each strip was mounted in a separate 15-ml bath containing Tyrode's solution with glucose (1 mg/ml) and maintained at 37 degrees C. All strips were equilibrated for 30 min with a gas mixture of 95% O2 and 5% CO2. At the end of this period of time, all strips were stimulated with field stimulation (FS) carbachol and KCl. After the last wash, the aeration was changed to hypoxic mixture (nitrogen-CO2) without glucose. At the end of 2 hr, the aeration was changed back to the normal 95% O2 and 5% CO2, and glucose was added to the buffer. After 1 hr of re-oxygenation, a second set of stimulations was performed. In order to represent hyperreflexia, the strips were stimulated at 32-Hz FS at 5-min intervals during the hypoxic period in half of the in vitro experiments. RESULTS: The results showed that Kohki Tea pretreatment protected the bladder's response to FS from the detrimental effects of repetitive stimulation and the detrimental effects of both in vitro ischemia and repetitive stimulation on the contractile responses to carbachol and KCl. CONCLUSIONS: These data are consistent with the concept that Kohki Tea acts by protecting the bladder from cellular damage caused by hypoxia and the generation of free radicals.

Administration, Oral↗

Focal hypoxia of the obstructed rabbit bladder wall correlates with intermediate decompensation.

AIMS: We showed that partial obstruction of the rabbit bladder outlet caused decreases in detrusor blood flow that were directly proportional to the level of decompensation present. Bladder decompensation is characterized by decreases in detrusor contractility, mitochondrial function, and sarco/endoplasmic reticulum calcium ATPase (SERCA) activity in obstructed rabbits. The current study was designed to create bladder decompensation and to relate its characteristic dysfunctions to the presence or absence of hypoxia in the obstructed rabbit bladder wall. Tissue hypoxia was visualized immunohistochemically after administration of a hypoxia probe in vivo. METHODS: Twelve New Zealand White rabbits were separated into two groups. The rabbits in group 1 received sham operations; the rabbits in group 2 received partial outlet obstructions by standard methods. Four weeks after surgery, each rabbit received an intraperitoneal injection of aqueous Hypoxyprobe-1, which forms protein adducts in cells having O(2) concentrations less than 14 microM. Two hours after injection, the rabbit was anesthetized and the bladder exposed through a midline incision. One full-thickness bladder strip was cut and immediately placed in fixative for immunohistochemical recognition and visualization of Hypoxyprobe-1-protein adducts. The remaining bladder was then excised, and three additional strips were cut for contractility studies. The remainder of the bladder was frozen for biochemical and slot-blot analyses. RESULTS: Bladder weight was increased fourfold after obstruction, and significant contractile and biochemical dysfunctions were observed that indicated an intermediate level of decompensation. Immunohistochemical visualization revealed focal areas of moderate to severe hypoxia in the detrusor smooth muscle (SM) and subserosal regions of these bladders. No hypoxia was observed in the obstructed bladder mucosa, consistent with the absence of biochemical dysfunction in this compartment, or in unobstructed bladders. Slot-blot analyses confirmed the presence of significant Hypoxyprobe-1-protein adducts in the detrusor of the obstructed bladder, whereas none were present in the control bladder detrusors. CONCLUSIONS: Partial outlet obstruction of rabbit bladders resulted in focal areas of moderate to severe hypoxia in the detrusor SM and subserosal regions concomitant with increased bladder mass, decreased contractile function, and selective metabolic dysfunctions of the SM consistent with an intermediate stage of decompensation. The metabolic characteristics of the normoxic mucosa were normal a were those of unobstructed bladders.

Adenosine Triphosphate↗

Effectiveness of vaginally administered oxybutynin on rabbit bladder function.

OBJECTIVES: To demonstrate the effectiveness of vaginally delivered oxybutynin on bladder function. Oxybutynin has been used for treatment of urge urinary incontinence for more than 20 years. METHODS: Thirty female New Zealand White rabbits were used for this experiment. Each rabbit was anesthetized, and the carotid artery was cannulated for blood pressure monitoring. The bladder dome was catheterized for both monitoring of bladder pressure and cystometry. After initial cystometry, acetylcholine (ACh) was injected into the vesical artery at 15-minute intervals for 4 hours. Cystometry was performed at the 2-hour mark and at the end of each experiment (4 hours). After the third ACh administration, vaginal or oral oxybutynin was given and the effect on the response to ACh, blood pressure, and cystometry was observed. RESULTS: The high dose of oxybutynin completely inhibited the response to ACh and significantly inhibited the micturition reflex. A 45% inhibition of micturition pressure occurred at the intermediate dose. Little effect on compliance after the low dose and a significant increase in compliance after the mid-dose occurred. Both the low and intermediate doses of oxybutynin reduced the response to ACh to approximately 50% of control. No blood pressure effects of vaginal oxybutynin were noted for any concentration. Oral oxybutynin showed very similar effects to that of the intermediate vaginal dose. CONCLUSIONS: Vaginally delivered oxybutynin was effective in decreasing bladder compliance and inhibiting intra-arterial ACh-stimulated bladder contractions with little or no effect on the vascular effect of ACh.

Acetylcholine↗

The effect of vitamin E on the response of rabbit bladder smooth muscle to hydrogen peroxide.

There is increasing evidence that ischemia, reperfusion, and the generation of free radicals are major etiological factors in the progression of bladder dysfunction after partial outlet obstruction. In vitro studies demonstrated that the magnitude of contractile dysfunction following exposure of bladder smooth muscle to hypoxia followed by re-oxygenation was related to the level of lipid peroxidation indicating that membrane lipid peroxidation participated in the contractile failure induced. Recent studies demonstrated that incubation of isolated strips of bladder smooth muscle with hydrogen peroxide (H2O2) result in progressive contractile dysfunctions and is associated with progressive increases in MDA (peroxidation product). The current study investigates if feeding rabbits a diet high in vitamin E protects the bladder from the effects of in vitro H2O2. Sixty-four male New Zealand White rabbits were separated into two groups: The rabbits in group 1 were fed a normal diet (28 rabbits) whereas the rabbits in group 2 were placed on a diet enriched with alpha-tocopherol (36 rabbits). After 3 weeks on the normal or high E diet, each rabbit was anesthetized and the bladder excised and cut into 6 isolated strips of bladder detrusor. Each strip was mounted in individual 15 ml baths containing oxygenated Tyrode's solution. The contractile responses to field stimulation (FS), carbachol. and KCl were determined. The strips were washed and exposed to one of the following concentrations of hydrogen peroxide (H2O2): 0% (control), 0.0625, 0.125, 0.25, 0.5, 1.0 and 3.0% for a period of 1 h. At the end of the hour each strip was washed free of H2O2 and a second set of contractile responses were performed and compared to the first set. At the end of the experiment, each strip was frozen and stored at -70 degrees C for analysis of malondialdehyde (MDA) as a measure of peroxidation. In both groups, H2O2 produced similar dose dependent decreases in the contractile responses to all forms of stimulation. In the normal-diet group H2O2 produced a dose dependent increase in MDA formation, whereas in the high E group there were no increases in MDA at any concentration of H2O2. Feeding rabbits a diet high in vitamin E protected the bladder smooth muscle from peroxidation, but had no significant effect on the contractile dysfunctions mediated by direct incubation with H2O2.

Animal Feed↗

Effect of age on hydrogen peroxide mediated contraction damage in the male rat bladder.

PURPOSE: Recent studies introduced the concept that reactive oxygen species (ROS) may be a major factor in the progressive deterioration of bladder function induced by benign prostatic hyperplasia in men and following partial outlet obstruction in animals. We determined whether bladder contraction sensitivity to ROS changes with aging. Using H2O2 to simulate ROS damage we compared the sensitivity of the contractile responses of bladder smooth muscle isolated from young and elderly rats to H2O2. MATERIALS AND METHODS: Male 3-month-old (young) and 12-month-old (elderly) Sprague-Dawley rats were used for this study with 24 per group. Each rat was anesthetized and the bladder was excised. Two longitudinal strips were cut from the bladder body. Each strip was placed in individual 15 ml baths containing oxygenated Tyrode's solution at 37C. Each strip was stimulated at 32 Hz for 20 seconds with pulses 1 millisecond in duration at 80 V. After electrical field stimulation (EFS) the response to 20 microM carbachol and 120 mM KCl was determined. The bath solution was then exchanged for H2O2 solutions of 6 different concentrations including 0%, 0.0625%, 0.125%, 0.25%, 0.5% and 1.0%. After 1 hour of exposure to H2O2 the tissues were washed free of H2O2, and the response to EFS, carbachol and KCl were measured again. At the end of the experiment the strips were weighed and frozen at -70C for malondialdehyde analysis. RESULTS: The magnitude of the contractile responses of the young and elderly rats to all forms of stimulation were equal. Hydrogen peroxide caused a dose dependent decrease in the contractile responses of bladder strips to all forms of stimulation. Contractile responses to carbachol and KCl were more sensitive to H2O2 than to EFS. Contractile responses of bladder strips isolated from elderly rats were significantly more sensitive to H2O2 damage than strips isolated from young rats. Malondialdehyde generation of bladder strips isolated from elderly rats was significantly greater than those from young rats. CONCLUSIONS: It is suggested that aging increases the sensitivity of detrusor contraction to oxidative damage.

Age Factors↗

Effect of doxazosin on rat urinary bladder function after partial outlet obstruction.

Hypoxia induced by partial outlet obstruction is believed to play a major role in both the hypertrophic and degenerative effects of partial outlet obstruction. Doxazosin (dox) is a clinically effective alpha-adrenergic antagonist used in the treatment of symptomatic benign prostatic hyperplasia (BPH). Although the major therapeutic effect of the agent is believed to occur on the smooth muscle components of the prostate by reducing prostatic urethral resistance and thus improving emptying, dox may have part of its clinical action via effects mediated by other actions, including via spinal alpha-adrenergic receptors or direct effects on the bladder, possibly via inhibition of vascular alpha receptors. The specific aim of the current study was to determine whether dox pretreatment on rats affects blood flow to the bladder and reduces the level of bladder dysfunction induced by partial outlet obstruction. In part 1, eight rats were separated into two groups of four rats each. Group 1 received oral administration of dox (30 mg/kg) for 4 weeks; group 2 received vehicle (5% dimethyl sulfoxide). After 4 weeks of treatment, blood flow studies were performed using fluorescent microspheres and the bladders excised, frozen, and submitted to Interactive Medical Technologies (IMT) for blood flow analysis. In part 2, 32 adult male rats were separated into four groups of eight rats each. Groups 1 and 2 received oral administration of dox (30 mg/kg) for 4 weeks, groups 3 and 4 received vehicle (5% dimethyl sulfoxide). At 4 weeks, the rats in groups 1 and 3 received partial outlet obstructions and treatment continued for an additional 2 weeks. After 6 weeks of treatment (total), each rat was anesthetized, the bladder excised, weighed, and isolated strips mounted and contractility studies performed. 1) Four weeks pretreatment of rats with dox increased blood flow to the bladder in both the control and obstructed groups. 2) Partial outlet obstruction induced a mild decrease in blood flow. 3) The magnitude of the increased bladder weight in the vehicle-treated obstructed group was significantly greater than in the dox-treated obstructed group. 4) Partial outlet obstruction resulted in significant decreases in the contractile response to field stimulation in both treated and non-treated rats. The magnitude of the decreased response was significantly greater in the non-treated rats. 5) The response to potassium chloride was significantly reduced by partial outlet obstruction in the vehicle-treated group but not in the dox-treated group. 6) The time to maximal tension was significantly increased in response to carbachol, adenosine triphosphate, and potassium chloride. However, the magnitude of the increase was significantly greater for the vehicle-treated obstructed groups stimulated by potassium chloride than for the dox-treated groups. Dox treatment of rats increased blood flow to the bladder and reduced the severity of the response to partial outlet obstruction. These beneficial effects would be due to pharmacological effects on alpha-adrenergic systems outside those present in the prostate. These include effects on blood flow to the bladder, effects on the micturition centers of the central nervous system, spinal reflexes, and alpha-adrenergic receptors in the urethra and bladder.

Adenosine Triphosphate↗

Effect of oral Kohki tea on bladder dysfunction induced by severe partial outlet obstruction.

PURPOSE: Extracts of the leaves of Engelhardtia chrysolepis, a subtropical plant that grows wild in southern China, have been used medicinally in east Asia for hundreds of years. A standard extract named Kohki tea (Maruzen Pharmaceuticals, Onomichi City, Japan) is sold over the counter in Japan as a sweet tea shown to confer many beneficial effects on general health and well-being. The tea contains strong antioxidants, including several dihydroflavonol glycosides. The results of previous studies show that natural products with antioxidant activities provide protective effects on the bladder of rabbits with partial outlet obstruction. We determined in vivo and in vitro whether oral pretreatment of rabbits with Kohki tea protects the bladder from dysfunction induced by partial outlet obstruction. MATERIALS AND METHODS: A total of 28 New Zealand White rabbits were separated into 4 groups of 7 each. Rabbits in groups 1 and 2 were treated by gavage with 100 mg./kg. Kohki tea daily in distilled water, while those in groups 3 and 4 were given distilled water. After 4 weeks of daily oral administration each rabbit was sedated, the bladder was catheterized and cystometry was performed at a filling rate of 1 ml. per minute. At the completion of cystometry the rabbits were immediately anesthetized. Moderate outlet obstruction was created in groups 1 and 3, and sham surgery was performed in groups 2 and 4. Treatment was continued for an additional 4 weeks, when each rabbit was sedated and cystometry was repeated. After cystometry the bladder was exposed through a midline incision, excised, weighed and 4 strips of bladder body were cut for contractility studies. The balance of the bladder was separated between smooth muscle and mucosa by blunt dissection, frozen in liquid nitrogen and stored at -70C for biochemical analyses. RESULTS: Partial outlet obstruction stimulated similar increases in the bladder weight of all obstructed rabbits. Partial outlet obstruction resulted in a significant decrease in bladder compliance in all obstructed animals. However, the bladder of obstructed rabbits given Kohki tea were significantly more compliant than those given water. Voiding pressures in the control group and the obstructed group given distilled water were approximately equal, while obstructed rabbits given Kohki tea showed significantly higher maximal voiding pressure. The contractile responses to all forms of stimulation were reduced by obstruction to a significantly greater degree in the rabbits not given tea than in those given tea. Sarcoplasmic reticulum Ca2+-adenosine triphosphatase enzyme activity of the bladder was significantly reduced in obstructed rabbits given vehicle but activity was not reduced in obstructed rabbits given Kohki tea. CONCLUSIONS: Kohki tea had a significant protective effect on bladder function, contractile responses and bladder biochemistry in rabbits with moderate to severe partial outlet obstruction.

Animals↗