Vascular network expansion, integrity of blood-brain interfaces, and cerebrospinal fluid cytokine concentration during postnatal development in the normal and jaundiced rat

Abstract

Background: Severe neonatal jaundice resulting from elevated levels of unconjugated bilirubin in the blood induces dramatic neurological impairment. Central oxidative stress and an inflammatory response have been associated with the pathophysiological mechanism. Cells forming the blood-brain barrier and the choroidal blood-CSF barrier are the first CNS cells exposed to increased plasma levels of unconjugated bilirubin. These barriers are key regulators of brain homeostasis and require active oxidative metabolism to fulfill their protective functions. The choroid plexus-CSF system is involved in neuroinflammatory processes. In this paper, we address the impact of neonatal hyperbilirubinemia on some aspects of brain barriers. We describe physiological changes in the neurovascular network, blood-brain/CSF barriers integrities, and CSF cytokine levels during the postnatal period in normobilirubinemic animals, and analyze these parameters in parallel in Gunn rats that are deficient in bilirubin catabolism and develop postnatal hyperbilirubinemia.

Methods: Gunn rats bearing a mutation in UGT1a genes were used. The neurovascular network was analyzed by immunofluorescence stereomicroscopy. The integrity of the barriers was evaluated by [¹⁴C]-sucrose permeability measurement. CSF cytokine levels were measured by multiplex immunoassay. The choroid plexus-CSF system response to an inflammatory challenge was assessed by enumerating CSF leukocytes.

Results: In normobilirubinemic animals, the neurovascular network expands postnatally and displays stage-specific regional variations in its complexity. Network expansion is not affected by hyperbilirubinemia. Permeability of the blood-brain and blood-CSF barriers to sucrose decreases between one- and 9-day-old animals, and does not differ between normobilirubinemic and hyperbilirubinemic rats. Cytokine profiles differ between CSF and plasma in all 1-, 9-, and 18-day-old animals. The CSF cytokine profile in 1-day-old animals is markedly different from that established in older animals. Hyperbilirubinemia perturbs these cytokine profiles only to a very limited extent, and reduces CSF immune cell infiltration triggered by systemic exposure to a bacterial lipopeptide.

Conclusion: The data highlight developmental specificities of the blood-brain barrier organization and of CSF cytokine content. They also indicate that a direct effect of bilirubin on the vascular system organization, brain barriers morphological integrity, and inflammatory response of the choroid plexus-CSF system is not involved in the alteration of brain functions induced by severe neonatal jaundice.

Keywords: Blood–CSF barrier; Blood–brain barrier; Cerebrospinal fluid; Choroid plexus; Cytokines; Gunn; Jaundice; Neurovascular; Postnatal development hyperbilirubinemia.

Fig. 1

Cerebral vascularization in normobilirubinemic and hyperbilirubinemic animals during post-natal development. A Typical immunohistological staining of the endothelial-specific RECA-1 protein on brain section, and corresponding transformed black and white image generated using ImageJ software and allowing to quantify the number of microvessel segments and the surface area per field of observation (1.42 mm² field); B Number of vessel segments per field in the cortex, cerebellum, pons and colliculi at three postnatal developmental stages in normobilirubinemic Nj animals. The data represent an average of measurements from 3 to 5 different locations (squares on the drawings) within each structure; C Comparative analysis of the number of vessel segments in several subregions of brain structures at the three different ages studied in normobilirubinemic Nj animals; D Comparative analysis of the number of vessel segments (left), and of vessel surface area (right), per field in three regions of the cerebellum (insert in B) between non-jaundiced and jaundiced 9-day-old animals. All data are expressed as means ± SD n = 4 animals (2 males, 2 females) for 9-day-old (P9) and 70-day-old rats (P70), and n = 6 animals (3 males, 3 females) for 18-day-old rats (P18), for each genotype. All fields had the same surface area of 1.42 mm². *, **, ***: statistically different values, p < 0.05, p < 0.01, p < 0.001, respectively, Anova followed by Tukey’s multiple comparisons test. For 9-day-old animals in C, only ** and *** significances are shown

Fig. 2

Blood–brain and blood–CSF barrier integrity in normobilirubinemic and hyperbilirubinemic male and female rats during post-natal development. The integrity of the barriers was assessed by measuring their permeability toward [¹⁴C]-sucrose used as a polar tracer. A Apparent Kin brain measured in 8-h-old animals. B True K_{in brain} measured in 9-day-old animals. C K_{in CSF} measured in 8-h-old (P0) and 9-day-old (P9) animals. Data are expressed as means ± SEM, individual values are shown as open circles. Note that P0 and P9 tissue data are expressed as apparent and true K_in, respectively. See methods for permeability constant definition and calculation

Fig. 3

Comparative analysis of cytokine levels in CSF and plasma of 1-day-old and 9-day-old wild-type animals. Data are expressed as pg/ml, mean ± SD of 4 and 8 samples from 1- and 9-day-old animals, respectively. *, **, ***: adjusted p < 0.05, p < 0.01, p < 0.001, respectively, paired t-test adjusted for multiple comparisons by controlling the False Discovery Rate set at 0.05. Cytokines whose mean did not exceed the lower limit of quantification in any group (Il-4, EGF) were not included in the statistical analysis. P1, P9: 1-, 9-day-old animals, respectively

Fig. 4

Developmental profiles of cytokines in the CSF of wild-type animals. Data are expressed as pg/ml (mean ± SD of 4, 8 and 9 samples from 1-, 9- and 17-day-old animals, respectively). *, **, ***: p < 0.05, p < 0.01, p < 0.001, respectively, Anova followed by Tukey’s multiple comparisons test. P values are indicated also for differences close to 0.05 significance. EGF was not detected at any stages. P1, P9, P17: 1-, 9-, 17-day-old animals, respectively. Genes depicted in blue are developmentally upregulated genes, those depicted in red are developmentally downregulated genes

Fig. 5

CSF cytokine content in normobilirubinemic and hyperbilirubinemic seventeen-day-old animals. Only cytokines for which differences between genotypes were statistically significant or close to significance are shown. Data are expressed as pg/ml, (Mean ± SD from 9 samples). *, **: p < 0.05 and p < 0.01, respectively, Anova followed by Tukey’s multiple comparisons test

Fig. 6

Neutrophile infiltration in CSF induced by P3C treatment in normobilirubinemic and hyperbilirubinemic developing rats. P3C was injected in 8-day-old animals and CSF sampled 14 h later. P3C induced a massive pleocytosis, and 91% of immune cells found in the CSF of Nj animals were PMNs. The pleocytosis was reduced in jj rats. Data are expressed as the number of PMNs counted in 1 µl of CSF, mean ± SEM, n = 9 (Nj) and 11 (jj) from 3 jj and 2 Nj litters. *, p < 0.05, one-tailed student’s t-test for unequal variance