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Purpose: To investigate histologically the influence of maternal caffeine exposure during pregnancy in vivo on crystalline lenses in neonatal rats.
Methods: Experimentally naive, female Wistar-albino rats (200–220 g) were mated with adult male rats over 2 days for copulation. After confirming pregnancy with a vaginal smear method, 50 gravid rats (dams) were randomly divided into five groups (n = 10 in each), consisting of one control and four experimental groups. Groups 1, 2 and 3 experimental dams were treated with intraperitoneal (i.p.) caffeine at doses of 25, 50 and 100 mg/kg/day, respectively, during pregnancy from gestational day 9 through to day 21. Group 4 dams were treated with caffeine in distilled water in a gavage at a dose of 50 mg/kg/day. Group 5 control dams were given i.p. saline solution daily for the same period. After normal delivery, the eyes were examined by slit-lamp biomicroscopy. The neonates were then killed by decapitation at postnatal days 1 or 30 and the eyes removed for histopathologic investigation of the lenses.
Results: Group 1 and control eyes had normal anterior lens capsules with a single layer of anterior cuboidal epithelial cells, regularly oriented cortical and nuclear lens fibres, and a clear posterior lens capsule with no lining epithelial cells behind the equator. In the remaining groups, histopathologic findings suggesting cataractogenesis included eosinophilic degeneration, lens fibre cell swelling and liquefaction, central lens fibres with retained nuclei, and prominent epithelial cells lining the posterior lens capsule behind the equator. Moreover, some lenses in group 3 had immature cataract on slit-lamp biomicroscopic examination at postnatal day 30.
Conclusion: Excessive maternal caffeine exposure during pregnancy had cataractogenic effects on developing crystalline lenses in newborn rat eyes, both macroscopically and histopathologically. If an appropriate dose of caffeine can be identified, caffeine-induced cataract formation may be used as a new experimental cataract model in animal studies.
Chemically speaking, 14C-caffeine is an alkaloid (1,3,7-trimethyl [2–14C] xanthine) with three methyl groups. It is the most frequently consumed stimulant among pregnant women and is present in many popular caffeinated beverages, foods and over-the-counter medications (Graham 1978). It crosses the umbilical cord and placenta easily and appears in the urine and plasma of neonates. Therefore, fetal levels are believed to be in equilibrium with maternal concentrations (Aldridge et al. 1981) and a given in-utero caffeine dosage may affect fetuses more than adults (Knutti et al. 1982).
While a biological causal mechanism has not been clearly delineated, there is a general consensus among toxicologists concerning the teratogenic response to maternal caffeine exposure during pregnancy in laboratory mammals, mainly rodents (Jiritano et al. 1985). Although some effects of caffeine relate to sleep, haemodynamics, motility, learning ability and anxiety in neonates (Devoe et al. 1993), teratogenic effects have been reported, including cleft palate, limb malformation and ectrodactyly (Scott 1983), craniofacial malformations (Wilson & Scott 1984), intrauterine growth retardation with preterm birth (Fortier et al. 1993), decrease in osteocytes (Wink et al. 1996), and even abortion with fetal loss (Infante-Rivard et al. 1993). In addition, it has been demonstrated that 91% of embryos treated with 25 mg/kg caffeine have regions of open neural tube compared with controls (14%) (Wilkinson & Pollard 1994).
In animal studies, caffeine administration leads to ocular hypertension, changes in the ciliary epithelium, and a decrease in the light sensitivity threshold as determined by campimetry (Higginbotham et al. 1989; Okimi et al. 1991; Kurata et al. 1997; Ovanesov 1998; Arushanian & Ovanesov 1999; Avisar et al. 2002). In addition, caffeine affects the accommodative function of the eye (Zhai et al. 1993), increases blood vessel resistance, and decreases blood flow in the human optic nerve head and choroid-retina (Okuno et al. 2002). Pregnant mothers expose their infants to caffeine with numerous detrimental and teratogenic effects. Caffeine stimulates the heart, respiratory system and central nervous systems, and the occurrence of fetal loss is characterized by a linear trend on the log scale for each 100 mg ingested daily during pregnancy (Infante-Rivard et al. 1993). Indeed, Matsuoka et al. 1987) demonstrated dose-dependent teratogenic effects of caffeine on the fetal heart, detectable at relatively low concentrations. The most susceptible stage is during septation of the heart, and some extracardiovascular anomalies have been demonstrated with skeletal malformations in some fetuses.
Congenital cataracts are responsible for up to 40% of blindness in children and the aetiology is still unclear (Ojofeitimi et al. 1999). Although it is biologically plausible that caffeine consumption can adversely affect the outcome of pregnancy, the epidemiological and experimental evidences are still variable, although the strongest evidence is for an effect on intrauterine growth and development (Fortier et al. 1993; Nehlig & Debry 1994). Because the half-life of caffeine is protracted more than four times longer than the normal 2.5 hours during which there is a rise in blood caffeine concentrations, the metabolism and elimination of caffeine from the blood are substantially delayed among pregnant women during the course of the second and third trimesters, and a given dosage affects neonates more than adults (Knutti et al. 1981). In addition, caffeine clearance from the body continues essentially unchanged during the first trimester of pregnancy. Moreover, human neonates have low levels of the enzymes needed to metabolize caffeine (Jiritano et al. 1985). Furthermore, the disposal of caffeine in the fetus and newborn is very slow. Therefore, high caffeine intake may result in heightened fetal exposure, with possible detrimental effects.
The increase in the rate of malformations has been demonstrated in rats given caffeine at a dose of 100 mg/kg/day or more (Thayer & Palm 1975). This effect is not seen at a dose of 50 mg/kg/day, and humans ingest caffeine at substantially lower doses (Wilson & Scott 1984). It has been found that auditory startle is delayed in litters of rats exposed to 75 mg/kg/day of caffeine during pregnancy (West et al. 1986). Likewise, eye opening is delayed at 25, 50 and 75 mg/kg/day of caffeine. In female rats, vaginal opening is delayed at higher doses. In addition, there is a small but statistically significant increase in the risk of low birth weight babies and spontaneous abortion in pregnant women consuming more than 150 mg of caffeine per day (Fernandes et al. 1998).
Due to the large worldwide gestational consumption of caffeinated beverages (e.g. coffee, tea, cola) and foods (e.g. chocolate), it is important to establish whether caffeine is actually teratogenic on various tissues and organs, including the eyes, during embryogenesis. Therefore, this experimental study aimed for the first time to investigate whether maternal caffeine exposure during pregnancy had any effect on embryo-fetal lenticular development in neonatal rats, both macroscopically and histopathologically, at postnatal days 1 or 30.
Material and Methods
The ethics committee of Gaziantep University reviewed the study, and the experiments conformed to the Principles and Guidelines for the Use of Animals in Research, Testing, and Education issued by the Committee on Educational Programmes in Laboratory Animal Science (1991). Rats were obtained from the Medical Sciences Experimental Research Unit of the university.
Animal and tissue preparation
We used experimentally naive, adult female Wistar-albino rats (200–220 g). The rats were acclimated to caged laboratory conditions for 2 weeks. They were allowed to feed with standard pellets during the study and were housed in stainless steel cages in a temperature and humidity-controlled room with a 12-hour light/dark cycle (8 : 00–20 : 00 hours with lights on). Female rats were paired with adult male rats over 2 days for copulation, in the proportion of two females for every male animal. After confirming pregnancy by the vaginal smear method (Inaloz et al. 2000; Evereklioglu et al. 2003, 2004), 50 pregnant primipar rats (dams) were randomly divided into five equal groups (10 rats in each group), consisting of one control and four experimental groups.
The given dose and route of caffeine used in the present study were similar to those applied in previous experimental studies, from 25 mg/kg to 100 mg/kg body weight/day (Thayer & Palm 1975; Concannon et al. 1983; Wilson & Scott 1984; West et al. 1986). On day 9 of gestation, a day critical for the development of teratologic effects in this species (Kimmel et al. 1984; Roy et al. 1998; Inaloz et al. 2000), experimental dams were treated with intraperitoneal (i.p) caffeine (Sigma, St. Louis, MO, USA) at doses of 25 mg/kg/day (group 1, n = 10), 50 mg/kg/day (group 2, n = 10) and 100 mg/kg/day (group 3, n = 10) during pregnancy from 9 through to 20 days of gestation. Group 4 dams (n = 10) were treated with caffeine in distilled water in a gavage at a dose of 50 mg/kg/day for the same period. Control dams (group 5, n = 10) were injected with i.p. saline solution daily during pregnancy for the same period. During caffeine administration, all groups were examined daily using ultrasonography.
All dams were allowed to undergo normal delivery on gestation days 20 or 21. After slit-lamp biomicroscopic examination had been completed, half of the litters in each group were immediately killed by decapitation on postnatal day 1, after which the eyes were enucleated and examined for histopathologic characteristics. Because many congenital cataracts are known to develop in the first postnatal month, the other half of the litters in each group were left in their cages until postnatal day 30, and were raised by their biological mothers. On postnatal day 30, slit-lamp biomicroscopic examination was performed, and the neonates were then killed by decapitation to allow for histopathologic examination of the lenses as in the first evaluation.
The globes were separately numbered, fixed in a solution of 10% formaldehyde and prepared for histological evaluation. The tissues were then embedded in paraffin wax. Sections of 4–6 µm were obtained, mounted on slides and stained with haematoxylin-eosin for routine light microscopy (Ozkiris et al. 2004). The slides were histologically investigated by a physician who was masked to the treatment group. Multiple sectioning of paraffin blocks was performed to obtain the desired lens section with maximal anterior−posterior diameter cutting through the middle of the lens.
In the control group of neonatal rats, both slit-lamp biomicroscopic and histopathologic examinations of the crystalline lenses revealed normal findings, with a regularly shaped, single layer of anterior cuboidal epithelial cells, regularly arranged cortical and nuclear lens fibres, and only peripherally located lens fibre nuclei (Fig. 1). In addition, the posterior lens capsules were clear, without any lining epithelial cells behind the equator. Similar normal findings were also observed in the lenses of group 1 litters. In the remaining experimental groups of neonatal rats, histological examination demonstrated striking findings of cataractous changes. The most common findings concerned irregularly oriented lens fibres and the presence of a number of swollen cortical fibre cells both in the anterior and posterior lens cortexes with some liquefaction (Fig. 2). Additional conspicuous findings were the presence of prominent epithelial cells lining the posterior lens capsule behind the equator, and retention of the nuclei in the central lens fibres with some peripheral eosinophilic degeneration (Fig. 3). Furthermore, some litters in group 3, treated with a higher dose of caffeine, were found to have developed cataract on slit-lamp biomicroscopic examination at postnatal day 30 (Fig. 4). Taken together, 34% of the lenses in groups 2, 3 and 4 showed at least one of the aforementioned histopathologic and/or biomicroscopic cataractous changes.
Maternal toxicity was not observed in any group. There were no resorbed or stillborn fetuses during caffeine administration in any group, but a total of seven litters were miscarried from two dams in the group treated with 100 mg/kg/day of caffeine. In addition, there were no macroscopic ocular abnormalities in the newborn litters of experimental or control animals. However, we did not concentrate on the other possible physical and developmental changes in dams and offspring as these effects have been evaluated in detail by many previous studies and were outwith the scope of the present investigation.
In the present study, caffeine was given during the organogenesis period (9–20 days). Low birth weight and low crown−rump length were found to occur in the caffeine-treated groups in a dose-dependent manner (data not shown). In histological examination, the crystalline lenses have an anterior subcapsular epithelial monolayer that normally terminates at the lens equator in haematoxylin-eosin-stained preparations (Hepsen et al. 1997; Doganay et al. 2002). Therefore, the thin posterior lens capsule is normally clear, with no lining epithelial cells. The migration of the lens epithelial cells over the posterior lens capsule behind the equator results in the formation of posterior subcapsular cataract (Evereklioglu et al. 2003). This kind of change was clearly present in some of the lenses in the offspring of experimental groups treated with moderate and higher doses of caffeine. In addition, as the peripheral lens fibres become displaced towards the centre of the lens, their nuclei normally disintegrate so that the central lens area lacks nuclei. Teratogenic effects of congenital diseases, like rubella, are characterized by the persistence of the lens epithelial nuclei within the lens fibres in the deeper and central area (Klintworth & Garner 1994). This finding was also observed in the lenses of the experimental group treated with 100 mg/kg/day of caffeine. Therefore, lens programming may be damaged by toxic insult of caffeine if a mother consumes a high dose during pregnancy, causing cataractous development by the distribution of anomalies in the cellular components of developing crystalline lenses.
Degeneration, destruction and liquefaction of the cortical lens fibre cells result in the formation of cortical cataract (Klintworth & Eagle 1999). The formation of vacuoles, globules or clefts in the lens cortex is observed in its incipient stage. Then, interrupted and folded lens fibres and cortical clefts are filled with morgagnian globules. Degenerated lens cortex swells osmotically, and total cortical liquefaction with swollen lens fibre cells eventually ensues. Such histopathologic changes were demonstrated by Rogers & Grabowski (1984) on mirex-induced cataractogenesis in perinatal rats. In the present study, we also observed such histological findings with marked destruction and liquefaction of the cortical lens fibres in the anterior and posterior poles. In addition, there were quite a number of irregularly oriented swollen cortical lens fibres. These observations were consistent with the histopathologic findings of cortical cataract. Indeed, slit-lamp biomicroscopic examination demonstrated that some of the lenses in pups treated with 100 mg caffeine had immature cortical cataract at postnatal day 30.
Some possible mechanisms for the detrimental effects may be hypothesized. Caffeine increases cellular cyclic AMP (cAMP) by inhibition of phosphodiesterases. Therefore, the rise in cAMP may interfere with fetal cell growth and development (Weathersbee & Lodge 1977). Caffeine-induced vasoconstriction of the vessels during pregnancy causes a decrease in the blood flow of the placenta (Kirkinen et al. 1983), cerebrum (Mathew et al. 1983; Cameron et al. 1990), and mesenteric artery in young healthy subjects (Stubbs & Macdonald 1995) and neonates (Lane et al. 1999), with an increase in resistance in the coronary artery (Bottcher et al. 1995). In addition, Lotfi & Grunwald (1991) have demonstrated that oral administration of 200 mg of caffeine causes a 13% decrease in macular blood flow at 1 hour after administration, compared to baseline. Furthermore, Okuno et al. (2002) recently demonstrated that caffeine increases vessel resistance and decreases blood flow in the human optic nerve head and choroid-retina. Therefore, these findings suggest that decreased ocular blood flow may have contributed to the detrimental effect of caffeine by hypoxic-ischaemic necrosis of the nourished tissues, including the developing crystalline lenses. Indeed, materno-fetal ischaemic vasoconstriction leading to malformations may occur even in moderate doses if caffeine is taken with alcohol, tobacco or the other vasoconstrictive or antimigraine medications. As a result, the US Food and Drug Administration has issued warnings regarding the use of caffeine during pregnancy, as its intake at this stage is associated with an increased risk of fetal loss (Food and Drug Administration 1980).
In conclusion, the present study demonstrated that caffeine treatment of pregnant rats induced histopathologic lens changes. The most significant ocular changes were observed in the litters of dams treated with 100 mg/kg/day of caffeine. The results may suggest that pregnant women should consume caffeine in high quantity with caution. This advice may also be appropriate for nursing mothers. More studies are needed to assess the long term effects of caffeine on surviving litters. Caffeine-induced cataract is an additional model for the study of cataract formation.