Body size and brain volumetry in the rat following prolonged morphine administration in infancy and adulthood

Abstract

Background: Prolonged morphine treatment in infancy is associated with a high incidence of opioid tolerance and dependence, but our knowledge of the long-term consequences of this treatment is sparse. Using a rodent model, we examined the (1) short- and (2) long-term effects of prolonged morphine administration in infancy on body weight and brain volume, and (3) we evaluated if subsequent dosing in adulthood poses an increased brain vulnerability.

Methods: Newborn rats received subcutaneous injections of either morphine or equal volume of saline twice daily for the first two weeks of life. In adulthood, animals received an additional two weeks of saline or morphine injections before undergoing structural brain MRI. After completion of treatment, structural T2-weigthed MRI images were acquired on a 7 T preclinical scanner (Bruker) using a RARE FSE sequence. Total and regional brain volumes were manually extracted from the MRI images using ITK-SNAP (v.3.6). Regions of interest included the brainstem, the cerebellum, as well as the forebrain and its components: the cerebral cortex, hippocampus, and deep gray matter (including basal ganglia, thalamus, hypothalamus, ventral tegmental area). Absolute (cm³) and normalized (as % total brain volume) values were compared using a one-way ANOVA with Tukey HSD post-hoc test.

Results: Prolonged morphine administration in infancy was associated with lower body weight and globally smaller brain volumes, which was not different between the sexes. In adulthood, females had lower body weights than males, but no difference was observed in brain volumes between treatment groups. Our results are suggestive of no long-term effect of prolonged morphine treatment in infancy with respect to body weight and brain size in either sex. Interestingly, prolonged morphine administration in adulthood was associated with smaller brain volumes that differed by sex only in case of previous exposure to morphine in infancy. Specifically, we report significantly smaller total brain volume of female rats on account of decreased volumes of forebrain and cortex.

Conclusions: Our study provides insight into the short- and long-term consequences of prolonged morphine administration in an infant rat model and suggests brain vulnerability to subsequent exposure in adulthood that might differ with sex.

Keywords: brain volume; infant; magnetic resonace imaging (MRI); neonatal; opioid; segmentation.

Figure 1

Pharmacological groups. Infant rat pups received treatment (Rx) with either morphine [morphine pups (MP); 10 mg/kg subcutaneously twice daily] or equivalent volume of saline [saline pups (SP)] starting on postnatal day (PD)1 for 2 weeks. One set of animals underwent brain MRI scan under light anesthesia on PD14 (A). Morphine-treated infant rats from another set underwent a 10-day weaning period from PD15-PD25 (gray area) and were allowed to growth to young adulthood when they received additional two weeks of treatment from PD56-PD75. The four adult groups were scanned after PD 57 (B) and included rats treated with saline in infancy and saline in adulthood (SSA); morphine in infancy and saline in adulthood (MSA); saline in infancy and morphine in adulthood (SMA); and morphine in infancy and adulthood (MMA).

Figure 2

Brain regions of interest. Figure illustrates representative infant rat brain with selected brain region masks in 3D view (A) and in sagittal section (B). Brain regions analyzed included brainstem (blue), cerebellum (CB; green) and forebrain. The latter was further sub-divided into cortex (CTX; red), deep gray matter (DGM; yellow), and hippocampus (HIPP; teal). A, anterior; D, dorsal (posterior); P, posterior (caudal), V, ventral (inferior).

Figure 3

Regional brain segmentation. Figure systematically outlines representative T2-weighted coronal sections with its 5 regional brain segmentations in an infant rat at 2 weeks of age. Coronal brain sections are organized from the most rostral (top left) to the most caudal (bottom right) ends. The most anterior section included the last section-containing cortex and excluded those that solely contained olfactory bulbs (Ob; not shown). Similarly, the most caudal section for the masking included the last tissue from the cerebellum (CB) and excluded more caudal sections solely with medulla (bottom coronal section without any masks). We used ITK-SNAP (v.3.6.0; www.itksnap.org) for regional brain segmentation that included cortex (CTX; red), deep gray matter (DGM; yellow), hippocampus (HIPP; teal), cerebellum (green), and brainstem (BS; blue). DGM included all deep structures of the forebrain (e.g., basal ganglia (BG), thalamus (TH), hypothalamus (HY), ventral tegmental area (VTA). D, dorsal (posterior); IC, inferior colliculus; P, posterior (caudal), SC, superior colliculus; V, ventral (inferior).

Figure 4

Average body weight at brain MRI scan. Graphs represent average body weight at brain MRI scan in grams (g) for infant (A) and adult rats (B,C). Infant rats pups were treated either with saline [saline pup (SP)] or morphine [morphine pup (MP)] twice daily for 2 weeks, which did not lead to weight differences in sex/group (SP (F vs. M), F(1,9) = 0.2; p = 0.66; MP (F vs. M), F(1,10) = 0.13; p = 0.73). Infant rats treated with morphine were smaller than those treated with saline [F(1,21) = 70.7, p < 0.001] without sex differences (A). Separate group of animals subsequently received additional 2 weeks of treatment in adulthood comprising total of 4 pharmacological groups (see also Figure 1): saline in infancy and saline in adulthood (SSA); morphine in infancy and saline in adulthood (MSA); saline in infancy and morphine in adulthood (SMA); and morphine in infancy and morphine in adulthood (MMA). Due to obvious sex differences in weight in adulthood, average weight of adult rats is separated by sex (B,C). Although trend in average body weight per pharmacological group was similar, group differences were found for male F(3,15) = 4.9, p = 0.015) but not female rats [F(3,23) = 1.1, p = 0.39]. ANOVA: *p < 0.05, **p < 0.01.

Figure 5

Immediate effects of prolonged morphine treatment on total and regional brain volumes in an infant Rat. Graphs show average absolute (cm³; A) and normalized brain volumes as a percent of total brain volume (%TBV; B) for the 2 groups of infant rats treated with saline [saline pup (SP); n = 11] or morphine [morphine pup (MP); n = 12] twice daily for 2 weeks. Absolute brain volumes of morphine-treated pups (MP) were smaller in comparison to saline-treated pups (SP) for total brain volume (TBV) and across all regions analyzed (A). No differences were observed in normalized brain volumes between groups (B). BS, brainstem; CB, cerebellum; CTX, cerebral cortex; DGM, deep gray matter; FB, forebrain; HIPP, hippocampus; ANOVA: *p < 0.05, **p < 0.01.

Figure 6

Long-term effects of prolonged morphine treatment in infancy on adult Rat total and regional brain volumes. Average absolute (cm³) and normalized brain volumes as a percent of total brain volume (%TBV) for female (A and A′, respectively) and male (B and B′, respectively) adult rats for the 2 groups of animals that underwent 2-week periods of treatment in infancy and adulthood: saline in infancy and saline in adulthood (SSA; n = 6 female; n = 5 male) and morphine in infancy and saline in adulthood (MSA; n = 7 female; n = 5 male). There were no significant differences in either absolute or normalized total or regional brain volumes between pharmacological groups for either sex using t-test. BS, brainstem; CB, cerebellum; CTX, cerebral cortex; DGM, deep gray matter; FB, forebrain; HIPP, hippocampus.

Figure 7

Total and regional brain volumes following prolonged morphine administration in adult rats ± previous exposure to morphine in infancy. Average absolute (cm³) and normalized brain volumes as a percent of total brain volume (%TBV) for female (A and A′, respectively) and male (B and B′, respectively) adult rats for the 3 groups of animals that underwent 2-week periods of treatment in infancy and adulthood: saline in infancy and saline in adulthood (SSA; n = 6 female; n = 5 male), saline in infancy and morphine in adulthood (SMA; n = 6 female; n = 5 male), and morphine in infancy and morphine in adulthood (MMA; n = 8 female; n = 4 male). There were no significant differences in average absolute total or regional brain volumes for either of sex following prolonged morphine administration only in adulthood (SSA vs. SMA). However, prolonged morphine administration in adulthood was associated with smaller total brain volume of female rats due to decreased absolute volumes of the forebrain and cortex. Cerebellum volume differences in males [F(2, 11) = 5.49, p = 0.022] are not considered significant due to conservative p value to account for multiple comparisons (significance p < 0.017). Abbreviations: BS, brainstem; CB, cerebellum; CTX, cerebral cortex; DGM, deep gray matter; FB, forebrain; HIPP, hippocampus; TBV, total brain volume. ANOVA: *p < 0.05, **p < 0.01.