Main

P-glycoprotein (P-gp) is a membrane ATP binding cassette transporter present in many tissues such as the kidneys, liver, intestines, testes, and brain (1). Due to its strategic location on the luminal membrane of brain endothelial cells, it has been suggested that it plays an important role in limiting the entry of drugs into the brain (2,3). In brain endothelial cells, a portion of the P-gps is localized in caveolae and interacts with caveolin-1 (4,5). It has been shown that when these two proteins interact, P-gp activity is inhibited (6). However the localization of P-gp in caveolae and its interaction with caveolin-1 remains controversial (7) and could depend on the specific cell line and detergent used in caveolae purification methods (8,9). At present, no studies have looked at caveolin-1 expression at the developing blood–brain barrier (BBB) or how its expression may modulate P-gp function at the developing BBB.

P-gp at the BBB limits the entry of substrates found the blood from moving into the brain. These substrates have to move from blood into the brain endothelium in order to be effluxed by P-gp as the substrate-binding site is in the cytoplasmic portion of P-gp. Substrates can also originate from the brain tissue and move into the brain endothelium to be effluxed by P-gp into the blood. Morphine is commonly used for the management of mild to severe pain and different experimental approaches have demonstrated that P-gp influences the distribution of morphine across the BBB (10,11,12,13), and hence its analgesic, and adverse effects.

Neonates have been shown to have a heightened sensitivity to the central depressive effects of opioids compared to older infants and adults in both animal and human studies (14,15,16,17,18,19). Furthermore, recent studies have documented that the newborn infant is sensitive to the respiratory effects of morphine when the mother is breastfeeding while taking codeine (20,21). This may be attributed to the limited expression of P-gp at the BBB during the neonatal period leading to increased morphine concentration in the brain (14,15). P-gp has been detected as early as embryonic day 10.5 in the mouse brain and served as the earliest marker for endothelial cell differentiation during BBB development (22). P-gp gene expression in the mouse brain is low during late gestation and reaches adult levels by 3 wk of life (22,23,24).

In humans, P-gp immunoreactivity was detected as early as 8–12 wk gestation in the fetal brain with its prevalence and intensity progressively increased with advancing age (25,26,27). However, even at term, P-gp expression in the fetus is low compared to adults (25). In contrast, the ontogeny of P-gp in the developing human infant has not been previously studied.

The objective of this study was to quantify the ontogeny of P-gp in endothelial cells of the developing BBB. We hypothesized that P-gp expression would be low at birth and increase dramatically in the neonatal period, explaining why young infants are sensitive to the central effects of morphine during the first months of life compared to older infants and adults.

Results

The intensity of P-gp immunoreactivity progressively increased in the brain with increasing age ( Figure 1 ). The intensity of P-gp in adults was significantly higher compared to that at 20–26 wk gestation (GA 20–26 wk) (P = 0.0002), 36–40 wk gestation (GA 36–40 wk) (P = 0.0002), and in infants aged 0–3 mo (PNA 0–3 mo) (P = 0.0044). Furthermore, there was a more pronounced decrease in P-gp intensity at GA 20–26 wk (P = 0.0011), GA 36–40 wk (P = 0.0013), and PNA 0–3 mo (P = 0.0173) compared to at PNA 3–6 mo. P-gp expression reaches adults levels by 3–6 mo of age (P = 0.74).

Figure 1
figure 1

Intensity of P-gp at various developmental ages. Seven brain microvessels from each sample were selected and the intensity of P-gp was quantified. The intensity of P-gp progressively increased during development, reaching adult levels by 3–6 mo. Data expressed as mean ± SEM at each developmental age. P-gp intensity was compared using an one-way ANOVA followed by the Tukey’s multiple comparison test (*P < 0.05, **P < 0.01, †P < 0.001).

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Discussion

P-gp expression at the BBB plays an essential role in limiting xenobiotic exposure through active efflux from the central nervous system (CNS). This is the first study examining the ontogeny of P-gp in the human BBB in the fetal, infant, and adult period. Our results indicate that the P-gp immunostaining intensity was relatively low during the fetal and newborn period, but increases significantly with postnatal maturation. By PNA 3–6 mo of life, P-gp intensity in microvessel endothelial cells of the cortex approximated that in adults. Thus, the increased sensitivity to opioids in newborn infants compared to older infants may be attributable, at least in part, to the low expression of P-gp at birth and thereby allowing opioids to concentrate in the CNS.

P-gp immunoreactivity was detected in microvessels of the cortex as early as GA 20 wk suggesting that P-gp plays an important role in neuroprotection during fetal brain development, as has previously been shown in animal models (28). Previous studies in the human fetal brain detected P-gp in brain microvessels at crown-rump lengths corresponding to GA 17–24 wk, serving as an early marker of BBB development (26,27,29). Virgintino et al. (27) first detected P-gp as diffuse cytoplasmic labeling of the endothelial cells lining the cortical microvessels, which became linear and continuous on endothelial cell membranes at GA 22 wk. Since Virgintino et al. did not investigate the expression of P-gp in the developing human cortex at GA 20 wk, it is possible that P-gp may have already been expressed at the endothelial cells of the cortex microvessel before GA 22 wk. Similarly, the youngest human fetal brain in the study, GA 22 wk, was stained positive for P-gp (25).

P-gp immunoreactivity in human cortex was confined to brain microvessels. Levels of P-gp increase with advancing age but do not reach adult levels by term indicating further postnatal changes. By 3–6 mo of life, P-gp levels in the brain endothelial cells approximated those in the adults. This developmental pattern mimics what has been reported in the mouse brain (23). P-gp expression in the mouse brain was limited during late embryogenesis and the newborn period and increased remarkably with postnatal maturation reaching adult levels by day 21 of life (23). In humans, the intensity of P-gp immunostaining in the microvessel endothelial cell increased from GA 22 wk up to term but the P-gp intensity at term was still significantly lower when compared to adults (25).

Although the functional impact of normal developmental changes in BBB P-gp expression has not been thoroughly investigated, studies in adults have demonstrated that P-gp plays an active role in the barrier function by protecting the brain against the accumulation of toxic xenobiotics (2,30,31,32). Recently, it has been shown that developmental changes in BBB P-gp contribute to the accumulation of cyclosporine A, a P-gp substrate in the murine brain (33). Specifically, cyclosporine A accumulation within the brain was the highest in newborn mice and decreased with increasing postnatal P-gp expression (33). A study in guinea pig brain, which has a brain growth pattern that resembles human fetal development, showed that the expression of P-gp protein in the brain microvessels increases with advancing age (28,34). Hence, premature and full-term neonates may be at risk for CNS drug toxicity due to the limited BBB P-gp expression in the brain microvessels. Furthermore, neonates and young infants have been shown to be more sensitive to the central depressive effects of morphine compared to older children and adults in both animal and human studies (14,15,16,17,18,19). Taken together with the findings from the present study, differences in BBB P-gp expression during brain development most likely enhance the CNS passage into the brain. As P-gp expression reaches adult levels by age 3–6 mo, older infants have the capacity to efflux morphine from the brain before it has a chance to exert its depressant effect. There are possibly other mechanisms for the increased neonatal sensitivity to opioid such as slower clearance rate of morphine and other narcotics, enhanced intestinal absorption, and higher BBB permeability due to other mechanisms. These mechanisms should be explored in the future to understand the underlying mechanisms that may contribute to drug sensitivity during the neonatal period.

There are a number of limitations to the present study that should be acknowledged. Postmortem cortex samples were used in this study. P-gp immunostaining and intensity may be modulated by the conditions that proceed at the time or after death such as hypoxia following intrauterine death, and umbilical cord or placental infarction. Intrauterine death comprises more than half of all fetal deaths (7/12). P-gp expression in murine brains was increased following hypoxia-induced treatment (35). Thus hypoxia could have artificially increased the P-gp intensity in the fetal samples. P-gp immunostaining and intensity may also be modulated by the postmortem interval. However, the postmortem interval was unknown in this study. Information regarding time from death to tissue sampling, time from sampling to tissue fixation, and method of tissue fixation were not collected. Only cortex samples were used in this study. Future studies should investigate the ontogeny of P-gp in other brain regions and astrocytes. Moreover, genetic polymorphisms, which were not assessed in this cohort, have been shown to alter P-gp expression (36,37). Future studies should address the impact of genetic polymorphisms on P-gp-medicated drug transport in newborns.

A challenge with using postmortem material for immunofluorescence is tissue autofluorescence. Autofluorescence is caused by the fixation technique and the presence of fluorescent pigments in tissues. The brain has high levels of fluorescent pigment, lipofuscin. To reduce these levels, copper sulfate was applied to quench background fluorescence caused by lipofuscin (4).

Moreover, aldehyde-based fixation used to fix the brain samples forms crosslinks between proteins. This may result in inability of some protein epitopes to bind to respective antibodies. Antigen-retrieval via heated sodium citrated in conjunction with proteinase K digestion was used to to improve transporter epitope detection (5). This study was insufficiently powered to examine the contributions of age within the fetus–infant group, and the effects of gender differences.

In conclusion, BBB P-gp expression is incomplete in the newborn period but increases rapidly after birth and reaches adult levels by 3–6 mo. The limited BBB P-gp expression in newborns may allow drugs to concentrate in the brain, and as a consequence, lead to increased sensitivity to many drugs. Insight regarding the development of P-gp expression gained from this cohort furthers our understanding of the pharmacodynamics behind neonatal drug exposure.

Methods

Subjects and Samples

Autopsies of adults and fetuses delivered at Mount Sinai Hospital in Toronto, Ontario, Canada were reviewed to identify cases with no history of neurological pathology, no chromosomal anomalies, normal neurological examination and a formalin fixed paraffin-embedded postmortem cortex block available for study. Fetuses of 20–26 wk gestation (GA 20–26 wk) (n = 5) and 36–40 wk gestation (GA 36–40 wk) (n = 7), representing the late second trimester and second half of the third trimester, were identified between 2010 and 2012. The fetal age was estimated on the basis of the crown-rump length and/or pregnancy records (last menstrual cycle and assessments of fetal physical maturity). In addition, adult subjects (mean age 53.6 ± 6.5 y old) (n = 8) were identified between 2010 and 2012.

Autopsies of infants from the Department of Pathology at the London Health Science Center in London, Ontario, Canada were reviewed to identify cases with no history of neurological pathology, no chromosomal anomalies, a normal neuropathological exam and postmortem a formalin fixed paraffin-embedded postmortem cortex block. Infants aged 0–3 mo (PNA 0–3 mo) (n = 8) and 3–6 mo (PNA 3–6 mo) (n = 7) were identified between 2009 and 2012. The infant age was estimated on the basis of the pregnancy records and neonatal physical maturity. Table 1 summarizes the characteristics and causes of death from the autopsy reports. This study was approved by the Research Ethics Board of the Mount Sinai Hospital in Toronto, Canada, University of Toronto in Toronto, Ontario, Canada, University of Western Ontario in London, Ontario, Canada and the London Health Science Center in London, Ontario, Canada. All the Research Ethics Board granted a waiver of consent for next of kin in this study based on the following two necessary conditions: (i) There is a public interest in this research while protecting the privacy of individuals and (ii) There are adequate safeguards to protect the privacy of individuals. It was also considered that given the subject group was deceased individuals, contacting the families would cause distress. The data collected in this study were coded and analyzed anonymously. No personal identifiers were collected.

Table 1 Characteristics of fetal, infant, and adult samples
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Immunohistochemistry

Coronal sections of 5-μm thickness were cut from the formalin fixed paraffin embedded postmortem cortex blocks and mounted on positively charged microscopic slides. The samples were deparaffinized using xylene and rehydrated through a series of graded ethanol. The sections were rinsed (three times) with phosphate-buffered saline (PBS). The sections were incubated with 10 nmol/l sodium citrate with 0.05% Tween 20 (pH 6.0 at 97 °C) for 30 min. The sections were incubated at room temperature in ammonium chloride (50 nmol/l; pH 7.4) with PBS for 10 min. The sections were blocked in 5% normal goat serum (1 h; room temperature). Subsequently, the sections were incubated with the mouse monoclonal anti-P-gp antibody D-11 (diluted 1:50; Santa Cruz, Dallas, TX) and double immunolabeled with rabbit anti-laminin antibody (diluted 1:50; Sigma-Aldrich, Oakville, Ontario, Canada) overnight at 4 °C. After rinsing (five times) with PBS, the sections were incubated (1 h; room temperature) with Alexa Fluor 488 goat anti-mouse (diluted 1:1,000; Life Technologies, Burlington, Ontario, Canada) and Alexa Fluor 594 chicken anti-rabbit (diluted 1:1,000; Life Technologies) for P-gp and laminin detection, respectively. The sections were rinsed and incubated with 0.5 μg/ml 4′,6-diamidino-2-phenylindole (DAPI) (Life Technologies; 10 min; room temperature). Sections were rinsed with PBS and distilled water before being incubated in copper sulfate (1 mmol/l; pH 5.0; 30 min; room temperature). Sections were then rinsed with distilled water, PBS and mounted with 1,4-diazabicyclo[2.2.2]octane. A negative control section was prepared by omitting the mouse monoclonal anti-P-gp antibody. Results from the negative control confirmed the specificity of the antibody staining for P-gp.

Spinning Disc Confocal Microscopy and P-gp Quantification

Sections were viewed using the WaveFX Spinning Disc Confocal System by Quorum (Guelph, Ontario, Canada) with optimized Yokogawa CSU X1, Hamamatsu EM-CCD digital camera Image EM (C9100-13), and Leica DM16000B inverted research grade motorized microscope run by the Volocity 5.2.2 Acquisition software (Improvision, PerkinElmer, MA). On the tripled-immunolabeled section, a sequential scan procedure was applied during image acquisition of the three channels, Alexa Fluor 488 (excitation at 488ηm and detection range 500–535 ηm; green fluorescence), Alexa Fluor 594 (excitation at 594 ηm and detection at 617 ηm; red fluorescence) and DAPI (excitation at 385–400 ηm and detection range 450–470 ηm; blue fluorescence). All images were acquired using constant imaging parameters for each channel (green channel: exposure time- 489 ms, sensitivity- 255, power 90%; red channel: exposure time- 689 ms, sensitivity- 255, power- 90%, and blue channel: exposure time- 697 ms, sensitivity- 255, power-90%). Confocal images were taken at 1 μm intervals through the z-axis of the section covering in total 6 μm in depth. Images of 0.5 × 0.5 mm were recorded digitally and stored as TIFF files with the Volocity 5.2.2 Acquisition software (Improvision, PerkinElmer). Confocal imaging was performed at The Lunenfeld-Tanenbaum Research Institute in Mount Sinai Hospital, Toronto, Canada.

P-gp intensity was measured in seven brain microvessels from each section using Volocity Quantification 6.3 (Improvision, PerkinElmer). Brain microvessels for measurement were selected using the following criteria: (i) contained unbranched segments of at least 50 μm in length, (ii) appeared undamaged in transmitted light and fluorescence modes, and (iii) the diameter of the microvessel was between 6–10 μm. The selected microvessel was outlined manually using the region of interest tool to measure the average P-gp intensity of vessel. The fluorescence intensity was corrected for background. Slides were processed by one investigator. A second investigator viewed the images to ensure that the microvessels selected met these criteria. The level of intensity for each vessel measured by Volocity Quantification 6.3 correlated with P-gp expression; as P-gp was more expressed, the intensity measured increased.

Statistical Analysis

Statistical analysis was performed using SPSS (IBM, version 20, Somers, NY). The intensity of P-gp among the developmental age groups was compared using a one-way ANOVA followed by a Tukey’s multiple comparison test.

Author Contributions

J.L, L.E.K., and G.K. designed the study. J.L., L.E.K., P.S., and D.C. collected the samples. J.L., S.B., and M.I. collected the data. J.L., S.B., S.G.M., and G.K. analyzed the data. J.L. wrote the manuscript. All authors critically reviewed the manuscript and provided important intellectual content prior to submission.

Statement of Financial Support

This work was supported by the Canadian Institute for Health Research Grant FRN-84220 (S.G.M).

Disclosure

The authors have no conflict of interest to declare.