Topical Application Of Caffeine Research Articles

Acute Antioxidant and Cytoprotective Effects of Caffeine in the Model of Neurovascular Injury by Oxidative Stress

Caffeine is commonly used in the neonatal intensive care units for respiratory disorders, but the effects of this medication in the neonatal cerebral circulation remain largely unexplored. We used newborn pigs as a clinically relevant model of the neonatal human brain to investigate the effects of caffeine on cerebral arterioles and primary neurovascular cells. First, we examined dose‐dependent cerebrovascular and systemic effects of water‐soluble anhydrous caffeine in anesthetized newborn pigs equipped with closed cranial windows. Caffeine administered systemically at therapeutic concentrations (20‐40 mg/kg i.v.) had no significant effects on diameter of pial arterioles, mean arterial blood pressure (MABP), and heart rate within 10‐60 min after the injection. When used at a higher concentration (70 mg/kg i.v.), caffeine produced dilation of pial arterioles (15‐20%) accompanied by an increased heart rate, without any effect on MABP. Topical application of caffeine (10‐5‐10‐3M) under the cranial window produced a dose‐dependent dilation of pial cerebral arterioles (15‐30%) that was fully reversed by a brief flushing with artificial cerebrospinal fluid. These in vivo data indicate that cerebral vascular cells are potential targets for caffeine. Next, we tested the hypothesis that caffeine exhibits cytoprotective properties in cerebral circulation during oxidative stress conditions. We used an in vitro model of acute oxidative stress‐induced neurovascular injury caused by excitotoxic and inflammatory conditions concomitant with epileptic seizures, asphyxia, and cerebral ischemia. Endothelial cells and cortical astrocytes are the key components of the neurovascular unit that are most vulnerable to oxidative stress injury. Primary cultures of cerebral vascular endothelial cells (CVEC) and cortical astrocytes from newborn pigs responded to pro‐inflammatory cytokine TNF‐alpha (30 ng/ml) and excitotoxic glutamate (1‐2 mM) by a surge in reactive oxygen species (ROS) production leading to apoptosis. NADPH oxidase is the major ROS‐generating system in the neurovascular unit during inflammation and excitotoxicity. We examined the acute effects of caffeine on oxidative stress‐induced neurovascular cell death caused by TNF‐alpha and excitotoxic glutamate. Caffeine (0.1‐10 µM) inhibited NADPH oxidase activation, reduced overall ROS generation, and attenuated major key events of apoptosis (caspase‐3 activation, DNA fragmentation, and loss of cell‐cell contacts) in CVEC and astrocytes exposed to TNF‐a and glutamate. These data demonstrate, for the first time, that caffeine exhibits acute antioxidant and cytoprotective effects in cerebral vascular endothelial cells and astrocytes. Overall, endothelial and astrocytic components of the neurovascular unit represent target cells for caffeine cytoprotective therapy in the neonatal cerebral circulation during oxidative stress.

PDE2 Is a Novel Target for Attenuating Tumor Formation in a Mouse Model of UVB-Induced Skin Carcinogenesis

Our previous studies demonstrated that the topical application of caffeine is a potent inhibitor of UVB-induced carcinogenesis and selectively increases apoptosis in tumors but not in non-tumor areas of the epidermis in mice that are at a high risk for developing skin cancer. While this effect is mainly through a p53 independent pathway, the mechanism by which caffeine inhibits skin tumor formation has not been fully elucidated. Since caffeine is a non-specific phosphodiesterase inhibitor, we investigated the effects of several PDE inhibitors on the formation of sunburn cells in mouse skin after an acute exposure to ultraviolet light B (UVB). The topical application of a PDE2 inhibitor, erythro-9-(2-hydroxy-3-nonyl) adenine hydrochloride (EHNA hydrochloride), stimulated epidermal apoptosis compared to control (P<0.01) and to a greater extent than caffeine whereas a PDE4 inhibitor attenuated the epidermal apoptosis compared to control (P<0.01). Since PDE2 hydrolyzes cyclic nucleotides, mainly cGMP, the effects of EHNA hydrochloride on epidermal apoptosis following UVB exposure may be mediated, in part, by increased cGMP signaling. Data demonstrated that the topical application of dibutyryl cGMP stimulated epidermal apoptosis (P<0.01) following an acute exposure to UVB. Treating UVB-pretreated mice topically with 3.1 µmole or 0.8 µmole of EHNA hydrochloride attenuated tumor formation to a greater extent than treating with 6.2 µmole caffeine when these compounds were applied once a day, five days a week for 18 weeks. These observations suggest a novel role for PDE2 in UVB-induced tumorigenesis and that PDE2 inhibitors that mediate cGMP signaling may be useful for the prevention and treatment of skin cancer.

Topical caffeine induces mydriasis in animals under ketamine‐xylaxine anesthesia

AbstractPurpose To determine the effect of topically applied caffeine on the pupil diameter during ketamine‐xylazine anesthesiaMethods Six‐week‐old Sprague‐Dawley rats were anesthetized with ketamine xylazine intraperitoneally. Caffeine, 72 mM, dissolved in vehicle, 9 mg/ml, hydroxypropylmethyl cellulose dissolved in water, or the vehicle only, was topically applied. Animals were kept under room light conditions. In experiment I, caffeine in vehicle was topically applied to one eye and vehicle only to the other eye in 10 animals. In experiment II, caffeine in vehicle was applied to both eyes in one group of 10 animals and vehicle only to another group of 10 animals. In both experiments, pupil diameter was measured on both eyes. Measurements were taken immediately after initiation of the anesthesia, and 10, 20, 40 and 60 min. later.Results In experiment I, vehicle only caused a slight transient increase of pupil diameter, peaking around 20 min. after initiation of anesthesia and then decreased below baseline. Caffeine in vehicle caused an initial fast decrease in pupil diameter and the decrease rate leveled out towards 60 min. after initiation of anesthesia. In experiment II, vehicle to both eyes induced a transient increase in pupil diameter that returned to baseline. Caffeine and vehicle on both eyes induced an exponentially declining decrease towards asymptote miosis. The miosis reversed to mydriasis after topical application of tropicamide 5 mg/ml.Conclusion Topically applied caffeine induces miosis in a ketamine‐xylazine anesthetized rat. There is some systemic effect on pupil diameter of the currently used topical application of caffeine. The miotic effect of topically applied caffeine is reversed by tropicamide.

Topically applied caffeine induces miosis in the ketamine/xylazine anesthetized rat

The aim of the present study was to examine if topically applied caffeine influences pupil size in ketamine/xylazine anesthetized animals. Two experiments were carried out. In the first experiment, caffeine was topically applied to one of the eyes of 10 ketamine/xylazine anesthetized animals, while vehicle only was topically applied to the contralateral eye. In the second experiment, caffeine was topically applied to both eyes in one group of 10 ketamine/xylazine anesthetized rats, while in another group both eyes vehicle only was topically applied to both eyes. In both experiments pupil diameter was measured at 0, 10, 20, 40 and 60 min after topical application. In three of the animals, the pupil was dilated with tropicamide 5 mg/ml at 60 min after the topical application of caffeine and the pupil diameter was measured. The first experiment showed a relative miosis in caffeine treated eyes as compared to the vehicle treated eye, that changed over time. The second experiment in line with the first experiment, also showed that topically applied caffeine causes a relative miosis as compared to vehicle only that changes over time. Eyes treated with caffeine reacted with quick dilatation after tropicamide application. Topical caffeine antagonizes ketamine/xylazine anesthesia induced mydriasis in a time dependent manner.

Pharmacokinetics for topically applied caffeine in the rat

Topically applied caffeine was recently identified as a promising candidate molecule for cataract prevention. Little is known about the pharmacokinetics for topically applied caffeine. Potential toxicity of 72 mM caffeine on the ocular surface and the lens was qualitatively monitored and no toxic effects were observed. The concentration of caffeine was measured in the lens and the blood after topical application of 72 mM caffeine to groups of 10 animals sacrificed at 30, 60, 90 and 120 min after topical application. The lens concentration decreased throughout the observation period while the blood concentration increased up to 120 min. Further, the concentration of caffeine in the lens and blood was measured 30 min after topical application of caffeine, the concentration of caffeine being 0.72, 3.34, 15.51 and 72 mM depending on group belonging, in groups of 10 animals. The caffeine concentration in lens and blood, respectively, increased proportionally to the caffeine concentration topically applied. The rat blood concentrations achieved were far below the equivalent threshold dose of FDA recommended daily dose for humans. This information is important for further development of caffeine eye drops for cataract prevention.

Abstract 569: Inverse relationship between p53 and phospho-Chk1 (Ser317) protein expression in UVB-induced skin tumors in SKH-1 mice.

Abstract Previous data demonstrated that the topical application of caffeine inhibited UVB-induced skin tumor formation. Studies showed that caffeine significantly diminished phospho (p)-Chk1 (Ser317) staining and increased the number of mitotic cells that expressed both cyclin B1 and caspase 3 in tumors, suggesting that caffeine induces apoptosis selectively in tumors by inhibiting the ATR/Chk1 pathway and promoting lethal mitosis. We also found that caffeine attenuated the UVB-induced decrease in mitotic cells with cyclin B1 to a greater extent in p53 knockout (KO) mice compared with p53 wild-type (WT) littermates. Therefore, we sought to investigate the mechanism by which caffeine could selectively sensitize p53-deficient cells to apoptosis following UVB exposure by inhibiting ATR/Chk1 pathway. Performing immunohistochemistry on stored tumor paraffin samples, we found an almost exclusive inverse relationship (&amp;gt;95%) between p53 expression and p-Chk1 (Ser317), but not p-Chk1 (Ser345), in all 42 keratoacanthomas and 11 squamous cell carcinomas examined. Tumors that expressed high levels and large areas of p53 protein had no detectable p-Chk1 (Ser317) expression, whereas tumors that expressed high levels and large areas of p-Chk1 (Ser317) protein had no detectable p53 expression. Squamous cell carcinomas that demonstrated heterogeneous p53 and p-Chk1 (Ser317) expression within the same tumor showed that the areas that expressed p53 were negative for p-Chk1 (Ser317) and the areas that expressed p-Chk1 (Ser317) were negative for p53. Similar patterns were observed for keratoacanthomas. Not only was the inverse relationship demonstrated in heterogeneous tumors that showed clusters of positive and negative cells, it was also demonstrated in heterogeneous tumors that showed mixtures of single cells either positive or negative for p53. These findings were consistent for the epidermis away from tumors that demonstrated no p-Chk1 (Ser317), but appreciable p53 protein in the basal layer. As expected, the inverse relationship could not be observed by western blotting tumors that expressed both proteins. However, tumors from p53 KO mice had elevated levels of p-Chk1 (Ser317) compared with tumors from p53 wild-type SKH-1 controls. A similar pattern was observed in normal epidermis from p53 KO mice after a single exposure to UVB, whereas p53 WT littermates induced p-Chk1 (Ser317), but to a much lesser extent. Moreover, we demonstrated that UVB-induced p-Chk1 (Ser317) was inhibited by caffeine in p53 KO epidermal cells in vitro. To our knowledge, this striking phenotypic negative association between two proteins has never been observed in skin tumors. These data illustrate the dynamic regulation of checkpoint function, suggesting that phosphorylation of Chk1 on Serine 317 is regulated by p53 status and that p53 may act as a molecular on and off switch for the phosphorylation of Chk1 on Serine 317. Citation Format: Jamie J. Bernard, You-Rong Lou, Sarah Peng, Tao Li, Paul Nghiem, Allan H. Conney, Yao-Ping Lu. Inverse relationship between p53 and phospho-Chk1 (Ser317) protein expression in UVB-induced skin tumors in SKH-1 mice. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 569. doi:10.1158/1538-7445.AM2013-569

Abstract 1217: Effects of topical applications of caffeine on phospho-Chk1 (Ser317), mitotic cells with cyclin B1 and mitotic cells with caspase 3 in skin tumors from UVB pretreated SKH-1 mice

Abstract In an earlier study we irradiated female SKH-1 mice with UVB (30 mJ/cm2) twice a week for 20 weeks and UVB treatment was stopped. Three weeks later, these tumor-free high-risk mice were treated topically with 100 µl of acetone or with caffeine (6.2 µmoles) in 100 µl of acetone once a day 5 days a week for 18 weeks. The results showed that topical applications of caffeine inhibited the formation of keratoacanthomas and squamous cell carcinomas by 44% and 72%, respectively. Caffeine treatment increased the number of caspase 3 (active form) positive cells in keratoacanthomas by 87% and in squamous cell carcinomas by 92%. These effects on apoptosis were selective for tumors since treatment of the mice with caffeine had little or no effect in non-tumor areas of the epidermis (Lu, Y-P et al: PNAS 99: 12455-12460, 2002). In the present study we further analyzed these stored paraffin blocks and found that some mitotic tumor cells are undergoing apoptosis (lethal mitosis) as indicated by staining for caspase 3. Caffeine treatment significantly increased the percentage of mitotic tumor cells with caspase 3 staining by 213% in keratoacanthomas, and by 212% in squamous cell carcinomas, when compared with acetone-treated control animals. In addition, caffeine treatment caused a 66% increase in the percentage of mitotic tumor cells with cyclin B1 staining in keratoacanthomas, which is associated with an increase in the percentage of mitotic tumor cells with caspase 3 staining. Immunohistochemical analysis with these tissue sections showed multiple focal areas of phospho-Chk1 (Ser317) - staining cells in 41% of keratoacanthomas and in 64% of squamous cell carcinomas, but there was none in non-tumor areas of the epidermis. The expression of phospho-Chk1 (Ser317) was almost exclusively in the cytoplasm of the tumor cells. Caffeine administration decreased the number of phospho-Chk1 (Ser317) positive tumors by 56% and the percentage of phospho-Chk1 (Ser317) positive cells by 64% in keratoacanthomas. Although there was little or no decrease in the percentage of phospho-Chk1 (Ser317) positive cells in squamous cell carcinomas, the intensity of staining was markedly decreased. Pearson's correlation coefficient indicated that there was a weak but statistically significant inverse relationship between the percentage of phospho-Chk1 (Ser317) positive cells and the percentage of caspase 3 positive cells in tumors. Our study suggests that topical application of caffeine to high-risk mice selectively enhances apoptosis in tumors by decreasing the level of phospho-Chk1 (Ser317), increasing the number of mitotic cells with cyclin B1 and by increasing the number of mitotic cells with apoptosis (caspase 3 staining) resulting in abrogation of the G2/M checkpoint and mitotic catastrophe (Supported by NIH Grants RO1 CA130857 and RO1 CA128997). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1217.

Protection from photodamage by topical application of caffeine after ultraviolet irradiation

Protection from photodamage by topical application of caffeine after ultraviolet irradiation

Protection from photodamage by topical application of caffeine after ultraviolet irradiation

Characterization of mechanisms that can reverse residual damage from prior skin exposure to ultraviolet (UV) would be of considerable biological and therapeutic interest. Topical caffeine application to mouse skin that had previously been treated with UV has been shown to inhibit the subsequent development of squamous cell carcinomas. We used an established mouse photodamage model to investigate other possible effects of topical caffeine application after UV. SKH-1 hairless mice were treated with ultraviolet B (UVB) followed immediately by topical application of caffeine or vehicle three times weekly for 11 weeks. Caffeine applied topically after UV treatment resulted in a significant decrease in UV-induced skin roughness/transverse rhytides as assessed by treatment-blinded examiners. Histologically, topical caffeine application after a single dose of UVB more than doubled the number of apoptotic keratinocytes as evaluated by sunburn cell formation, caspase 3 cleavage and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labelling (TUNEL) staining. A trend towards decreased solar elastosis was noted in the caffeine-treated group although this was not statistically significant. Other histological parameters including epidermal hyperplasia, solar elastosis and angiogenesis were increased in mice treated with UV but topical application of caffeine did not alter these particular UV effects. These findings support the concept that topical application of caffeine to mouse skin after UV irradiation promotes the deletion of DNA-damaged keratinocytes and may partially diminish photodamage as well as photocarcinogenesis.

Effect of administration of caffeine or green tea on the mutation profile in the p53 gene in early mutant p53-positive patches of epidermal cells induced by chronic UVB-irradiation of hairless SKH-1 mice

Irradiation of SKH-1 mice with UVB light for 20 weeks resulted in a large number of patches of epidermal cells, which was visualized with an antibody that recognizes mutated p53 protein. Oral treatment of mice with caffeine (0.4 mg/ml) or green tea (6 mg tea solids/ml) as the drinking fluid during UVB irradiation decreased the number of patches by approximately 40%. Sequencing analysis of the p53 gene (exons 3 to 9) detected 88, 82 or 39 point mutations in 67, 70 or 29 patches from water, caffeine or tea treated mice, respectively. A major hotspot at codon 270 (Arg-->Cys) accounted for 47.7% (water), 70.7% (caffeine) or 46.2% (tea) of all mutations. Patches from caffeine treated mice had fewer types of mutations than patches from mice treated with water or tea. Administration of caffeine or tea during 20 weeks of UVB irradiation eliminated mutations at codons 149 (Pro-->Ser) and 210 (Arg-->Cys) but increased the frequency of mutations at codon 238 (Ser-->Phe). Topical applications of caffeine (1.2 mg in 100 microl acetone) once a day, five times a week for 6 weeks after stopping UVB decreased the number of patches by 63% when compared with mice treated with acetone. DNA sequencing analysis detected 63 and 68 mutations in 48 and 57 patches from acetone or caffeine treated mice, respectively. Although no differences in the frequency, position or types of mutations were observed, the caffeine group harbored less homozygous mutations (12.3% of the total) than the acetone group (31.3% of the total, P = 0.029). In summary, oral treatment of mice with caffeine or green tea during chronic UVB irradiation changed the mutation profile of the p53 gene in early mutant p53 positive epidermal patches, and topical applications of caffeine after discontinuation of chronic UVB irradiation specifically eliminated patches harboring homozygous p53 mutations.

Topical applications of caffeine or (-)-epigallocatechin gallate (EGCG) inhibit carcinogenesis and selectively increase apoptosis in UVB-induced skin tumors in mice.

SKH-1 hairless mice were irradiated with ultraviolet B (UVB) twice weekly for 20 weeks. These tumor-free mice, which had a high risk of developing skin tumors during the next several months, were then treated topically with caffeine (6.2 micromol) or (-)-epigallocatechin gallate (EGCG; 6.5 micromol) once a day 5 days a week for 18 weeks in the absence of further treatment with UVB. Topical applications of caffeine to these mice decreased the number of nonmalignant and malignant skin tumors per mouse by 44% and 72%, respectively. Topical applications of EGCG decreased the number of nonmalignant and malignant tumors per mouse by 55% and 66%, respectively. Immunohistochemical analysis showed that topical applications of caffeine or EGCG increased apoptosis as measured by the number of caspase 3-positive cells in nonmalignant skin tumors by 87% or 72%, respectively, and in squamous cell carcinomas by 92% or 56%, respectively, but there was no effect on apoptosis in nontumor areas of the epidermis. Topical applications of caffeine or EGCG had a small inhibitory effect on proliferation in nonmalignant tumors as measured by BrdUrd labeling (16-22%), and there was also a similar, but nonsignificant, inhibitory effect on proliferation in malignant tumors. The results suggest a need for further studies to determine whether topical applications of caffeine or EGCG can inhibit sunlight-induced skin cancer in humans.

An in vivo model for studying the dynamics of intracellular free calcium changes in slow- and fast-twitch muscle fibres.

The understanding of the regulation of the free cytosolic [Ca2+] ([Ca2+]i) in skeletal muscle is hampered by the lack of techniques for quantifying free [Ca2+]i in muscle fibres in situ. We describe a model for studying the dynamics of free [Ca2+]i in the fast-twitch extensor digitorum longus (EDL) and the slow-twitch soleus (SOL) muscles of the rat in vivo using caffeine superfusion to induce changes in free [Ca2+]i. We assumed that differences in sensitivity between the two muscle types for this substance reflect differences in intracellular Ca2+ handling in the fibres of which these muscles consist. The Indo-1 ratiometric method, using intravital microscopy with incident light, was adapted to measure free [Ca2+]i in vivo. Fluorescence images were collected by means of a digital camera. Caffeine superfusion at 37 degrees C for 2 min, at concentrations of 1, 2, 5, 10 or 20 mmol/l, induced a concentration-dependent increase in free [Ca2+]i and revealed differences in caffeine sensitivity between the muscle types, with the SOL being more sensitive. In a separate set of experiments the contracture threshold, as assessed by topical application of caffeine, was determined in both muscle types. EDL had a higher threshold for developing contracture than SOL. These finding are in agreement with previous in vitro studies. We may conclude that the dynamics of free [Ca2+]i can be assessed reliably in intact mammalian muscle in vivo.