Elsevier

Neuroscience

Volume 285, 29 January 2015, Pages 128-138
Neuroscience

Effects of thyroxine treatment on histology and behavior using the methimazole model of congenital hypothyroidism in the rat

https://doi.org/10.1016/j.neuroscience.2014.11.021Get rights and content

Highlights

  • There is a therapeutic window for T4 replacement therapy in congenital hypothyroidism.

  • The sensitivity of behavioral assessments is necessary in this area of research.

  • T4 treatment by P14 is necessary to protect dendritic branching.

  • T4 treatment by P7 is necessary to protect NGF expression.

  • T4 replacement should be provided as early as possible for the treatment of CH.

Abstract

The timing of thyroxine (T4) replacement treatment in congenital hypothyroidism (CH) has been suggested to be important for optimizing cognitive recovery in humans; however this has not been fully established using modern animal models of CH. Consequently, the current studies investigated the ameliorating effects of postnatal T4 treatment on neuropathology and behavior in CH rats. Rat dams were administered methimazole to produce CH offspring, then brain tissue from male CH pups was analyzed to determine the effects of postnatal (P3, P7, P14 and P21) T4 treatment on hippocampal dendritic branching and the expression of nerve growth factor (NGF). Two operant behavioral procedures were employed to confirm and extend previous findings obtained using this model, and to investigate timelines for instigating T4 treatment on improved behavioral outcomes. T4 treatment initiated at P14 was protective of a reduction in dendritic branching in the hippocampus, and initiated at P7 was protective of a reduction of NGF expression in the fimbria of the hippocampus. Induction of CH did not affect the acquisition of simple operant response rules but had a significant effect on the acquisition of complex operant rules subsequently imposed. Furthermore, T4 treatment initiated at P3 protected learning deficits seen following the imposition of complex operant response rules. These findings indicate T4 treatment initiated at P7 is sufficient for the protection of hippocampal NGF expression and dendritic branching but for the protection of complex behavioral abilities T4 treatment is necessary prior to or approximating P3.

Introduction

Adult hypothyroidism resulting from under-activation of the thyroid gland was first described by Gull (1874). Subsequently, Kendall (1915) isolated thyroxine from the thyroid gland, and further research demonstrated that iodine is necessary for the synthesis of thyroxine (Harington and Barger, 1927). Iodine deficiency affects the thyroid gland (e.g., Chapman, 1941), the production of thyroxine is reduced in hypothyroidism (French and Vanwyk, 1964), and thyroxine deficiency results in hypothyroidism in humans (e.g., Pharoah et al., 1971). Areas of the world where iodine deficiency is prevalent are occupied by approximately one-third of the global population (Zimmerman, 2009). Thyroid hormone deficiency, present at the time of birth through low maternal iodine levels is a major contributing factor to congenital hypothyroidism (CH) in newborns. This condition is treatable if diagnosed early but results in physical and psychological abnormalities if diagnosed late (LaFranchi, 1999).

In their review, Rastogi and LaFranchi (2010) pointed out that before the instigation of screening programs in newborns the incidence of CH diagnosed after clinical manifestations was approximately 1 in 7000–10,000 in Sweden (Alm et al., 1978), although the global incidence varied widely according to geographical location. For example, the French newborn screening program reported the incidence of permanent hypothyroidism with an approximate similarity to that reported in Sweden, and found the incidence to be 1 in 10,000 (Gaudino et al., 2005). Whereas a report based on the Greek Cypriot population found the incidence of permanent hypothyroidism to be dramatically higher at 1 in 800 (Skordis et al., 2005), and in Iran the incidence of CH was found to be approximately 1 in 600 (Ordookhani et al., 2002, Iranpour et al., 2006). Obviously, in the two-thirds of the global population where iodine deficiency is not prevalent, and where basic statistics relative to CH are available, the data on incidences of CH vary widely from country to country and from society to society. Consequently, it is reasonable to assume that in the one third of the global population where iodine deficiency is prevalent, accurate data relative to incidences of CH might be more difficult to obtain, and the incidence of CH might be higher than the global averages currently reported.

There is a direct correlation between the delay in diagnosis and treatment of CH and the degree of physical and neurological degeneration and psychological dysfunction (Gruters and Krude, 2007). Also, the timing of thyroid hormone replacement treatment in humans appears to be essential for optimizing neurocognitive recovery. For example, Bongers-Schokking and de Munick Keizer-Scharma (2005) reported that a delay in serum thyroxine T4 (T4) normalization of over 1 week after birth can result in a significant reduction in intelligence scores. Selva et al. (2005) found that T4 normalization beyond 2 weeks of birth resulted in CH subjects scoring lower on cognitive tests than CH subjects whose T4 levels were normalized less than 2 weeks after birth. From a clinical perspective, cognitive deficiency, motor deficits, lethargy, and generally slowed metabolism have all been reported as characteristics of CH (Glorieux and La Vecchio, 1983, Hetzel et al., 1988, Fuggle et al., 1991, Stein et al., 1991, Kooistra et al., 1994, Sher et al., 1998). These observations from humans have also been mirrored in data collected employing non-human experimental animals.

Perinatal reductions in thyroid hormone have been used to produce animal models of CH (Pickering and Fisher, 1953, Richards et al., 1990, Biesiada et al., 1996, Piosik et al., 1996), and a modern method of modeling CH in experimental animals has been the administration of anti-thyroid drugs, such as methimazole (MMI). MMI acts by inhibiting the incorporation of iodide into thyroglobulin, the thyroid hormone precursor protein (Cooper, 1984, Cooper et al., 1984). MacNabb et al. (1999) administered MMI to rat dams and investigated the induced CH on behavior in the rat pups produced. Previous studies had investigated effects on the righting reflex (Comer and Norton, 1982, Rice et al., 1987, Weller et al., 1996), exploratory behavior (Morgan and Einon, 1976, Schalock et al., 1979, Tamasy et al., 1986a, Tamasy et al., 1986b), home orientation (Johanson et al., 1980), maze learning (Hendrich et al., 1984, Comer and Norton, 1985, Akaike et al., 1991), and avoidance learning (Schalock et al., 1977, Schalock et al., 1979, Henck et al., 1996). By employing a completely novel approach, MacNabb et al. (1999) investigated operant behavior based on lever-press switching rules in CH rats and found that throughout the acquisition of a series of fixed-ratio (FR) schedules of responding for food reinforcement (FR-1, FR-3, FR-5 and FR-10) there were no differences in the behavioral abilities of CH rats born of MMI-administered dams. However, under a condition where it was a requirement to choose the appropriate operant lever for responses resulting in food reinforcement, CH rats were severely impaired. That is, on having made a choice between two operant levers, after making an error the CH rats perseverated on the incorrect lever rather than switching to the correct lever.

In a follow up study, MacNabb et al. (2000) replicated their original findings and expanded these through the inclusion of additional experimental groups. In that study they included four groups of CH offspring that were given subcutaneous (s.c.) T4 injections (0.02 μg/g) beginning on postnatal day 1 (P1), P7, P13 or P19. They found that T4 treatment beginning on P1, P7 and P13 significantly improved operant performance, whereas T4 treatment beginning on P19 had no effect. This led them to conclude that the duration of the CH condition prior to T4 treatment had a direct influence on operant performance, and they reiterated the suggestion (LeClerc et al., 1985, Bernal and Nunez, 1995, Calikoglu et al., 1996) that the main effect of CH on neural development might be to reduce the amount of time in which the nervous system is exposed to agents co-ordinating developmental processes. Furthermore, MacNabb et al. (1999), in accordance with findings by Meza et al. (1996) and Uziel et al., 1985a, Uziel et al., 1985b, suggested it may be that a developmental ‘window’ is open for a shorter temporal duration as a result of CH. So, some elements within the brain might not have the time necessary for completion of normal neurodevelopmental consolidation.

Reid et al. (2007), employing a more basic behavioral technique than the operant approach reported by MacNabb et al. (1999) and MacNabb et al. (2000), used a water-maze procedure in an attempt to further investigate the concept of a window of therapeutic efficacy for T4 treatment following maternal MMI-induced CH in rat offspring. In their study, two groups of CH offspring received T4 treatment (0.02 μg/g, s.c.) commencing on P7 or commencing on P21. Reid et al. (2007) found that relative to behavioral performance, untreated CH produced an impairment of spatial learning and memory up to 98 days of age in the CH rats (the outer limit of their study), which supported the findings of previous research investigating neonatal hypothyroidism (Akaike et al., 1991), perinatal hypothyroidism (Comer and Norton, 1985), and CH in hyt/hyt mice (Anthony et al., 1993). Memory retention was significantly better in the CH group that received T4 treatment beginning at P7 but T4 treatment beginning at P21 had no effect. Reid et al. (2007) concluded that this added credence to empirical findings suggesting that for the alleviation of chronic behavioral impairments T4 treatment should be instigated during a critical stage postnatally, in the earlier (approximating P7) rather than the later (P21) stage of postnatal development.

The work reported here used the protocol of MMI administration to pregnant rat dams for the induction of CH in their offspring in an attempt to further investigate the window for effective T4 treatment in CH. T4 treatment was instigated at P3, P7, P14 and P21 for the purpose of determining the effects of CH and T4 treatment on dendritic branching in the hippocampus, an area of the brain directly implicated in learning and memory (e.g., Jerrard, 1993), and of determining the expression of nerve growth factor (NGF) in the fimbria of the hippocampus, an area of the brain associated with production of the neurotransmitter acetylcholine (e.g., O’Keefe and Nadel, 1978, Olton et al., 1979), known to be involved in learning and memory processes (Storm-Mathisen and Gulberg, 1974). Two complex operant behavior analytical procedures, a 30-s conjunctive schedule (e.g., Hernstein and Morse, 1958) and a delayed matching-to-sample (DMTS) schedule (e.g., Berryman et al., 1963) were used to investigate T4 treatment relative to operant performance.

The main findings of this study were that reduced dendritic branching in the hippocampus was protected by T4 treatment initiated at P14, and reduced NGF expression in the fimbria of the hippocampus was protected by T4 treatment initiated at P7. The acquisition of simple operant response rules was not affected by the induction of CH, however following the imposition of complex response rules, effects on learning were alleviated only as a result of T4 treatment initiated at P3.

Section snippets

Animal subjects and the induction of CH

For all of the experiments reported, to produce CH subjects Sprague–Dawley rat dams (Harlan, Hillcrest, UK) timed pregnant to embryonic day 13 were housed individually with food and water available ad libitum and were maintained at 23 °C under a 12-h light/12-h dark cycle (lights on at 0800 h). MMI (Sigma, Gillingham, UK) was administered via the drinking water (0.025%) to the pregnant dams from embryonic day 16 until P25 (e.g., MacNabb et al., 1999, MacNabb et al., 2000, Reid et al., 2007). This

Results

Dendritic branching was significantly reduced (Fig. 1; p < 0.05) in the hippocampus of CH rat pups that did not receive T4 treatment and in the CH group that received T4 treatment initiated at P21, as compared to the age-matched executive control group of normal rats and the CH groups that received T4 treatment initiated at P3, P7 and P14. Also, NGF expression was significantly reduced (Fig. 2; p < 0.05) in the fimbria of the hippocampus in CH rat pups that did not receive T4 treatment and in the

Discussion

This series of studies was designed to investigate the effects of temporal delays in T4 treatment to CH rats relative to neuropathological and behavioral outcomes. It has been proposed that CH impairs co-ordinated neurological developmental processes (Bernal and Nunez, 1995, Calikoglu et al., 1996, LeClerc et al., 1985), and it has been reported that CH decreases the numbers of dendrites, and increases abnormal dendritic morphology (Eayrs, 1955, Brown et al., 1976, Faivre et al., 1984, Ipifia

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