Ministry of Health of the Republic of Moldova State University of Medicine and Pharmacy “Nicolae Testemitanu” GENERAL MEDICINE FACULTY Department of… [602241]

Ministry of Health of the Republic of Moldova State University of Medicine and Pharmacy “Nicolae Testemitanu” GENERAL MEDICINE FACULTY Department of Pathophysiology and Clinical Pathophysiology DIPLOMA THESIS PATHOPHYSIOLOGY OF NEONATAL JAUNDICE
Student: [anonimizat] : Alexandrov Viacheslav Year,Group Nr: 6th year group 1353 ScienAfic coordinator Dr. Lilia Tacu Chisinau ,20181

SummeryI.IntroducPon………………………..……………………………………..…………………………………….. 1.1 GeneraliAes about the unconjugated hyperbilirubinemia………………………………. 1.2 The Aim of the study………………………………………………………………………………………… 1.3 ObjecAves………………………………………………………………….…………………………………….. 1.4 TheoreAcal importance and pracAcal value of the work……………………….………….. II. Literature review………………………………………………………………………………………………. 2.1 Heme and Bilirubin metabolism……………………………………………………………….………. 2.2 DescripAon and possible cause in elevaAon of unconjugated bilirubin……………… 2.3 The beneficial effect of unconjugated bilirubinemia………………………………………… 2.4 The hazardous effect of unconjugated bilirubin on CNS……………………………………. 2.4.1 The hazardous effect of unconjugated bilirubin on non neural cells…………. 2.4.2 The hazardous effect of unconjugated bilirubin on neural cells……………………… 2.5 Possible outcome complicaAon and treatment associaAon epilepsy……………….… Conclusion…………………………………………………………………………………………………………..…. Bibliography…………………………………………………………………………………………………….……..
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I.Introduction1.1 Generalities about the unconjugated hyperbilirubinemia. Slightly more than half of all neonates become visibly jaundiced in the first week of life. The accumulation of unconjugated bilirubin can be seen initially as yellow discoloration of the skin and sclera. The serum level required to cause jaundice varies with skin tone and body regions, the discoloration of the skin have head-to-foot direction, become visible on the sclera initially at a level of 2-3 mg/dL, on the face 4-5 mg/dL, umbilicus at 15 mg/dL, and at the feet at about 20 mg/dL. Almost all newborn infants develop a total serum of plasma bilirubin level greater than 1 mg/dL, which is the upper limit of normal for adults. Even that in most newborns its reflect normal transitional phenomenon, in some cases in may rise excessively and requires medical attention and hospital readmission. High levels of unconjugated bilirubin have neurotoxic effect and can cause death, lifelong neurological sequelae ranging from lack of coordination to severe motor abnormalities. The clinical manifestations of elevated unconjugated bilirubin vary in form and severity and influences by some general and personal factors such as degree of prematurity, stage of regional brain development, quantity, and duration of hyperbilirubinemia, genetic predisposition, hypoalbuminemia, sepsis and more. This thesis will describe initially the possible etiology of elevated bilirubin, the spectrum of beneficial and hazardous effects of elevated unconjugated hyperbilirubinemia by physiopathological evidence results from exposure. Additionally new researches shows new aspects of evidence with lifelong complication from commonly used method to reduce the bilirubin levels. Due to that, those clinical manifestation, spectrum of possible beneficial effect, range of hazardous effects, and long life complication after phototherapy, requires new point of view in evaluation, management , and possibly new protocols.
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1.2 The aim of the studythe aim of the present study is to present pathophysiological mechanisms of elevation in bilirubin levels in neonates and ongoing evident upon causes, manifestations of hazardous and beneficial effect of elevated bilirubin, as well possible long term complications associated with phototherapy treatment. 1.3 Objectivies 1.To determine possible physiological and pathological cause of elevation in bilirubin level in neonatal period.2.To determine pathophysiological mechanism of beneficial effect of elevated bilirubin. 3.To determine the pathophysiological mechanism in hazardous effect of elevated bilirubin in neonatal 4.To determine possible long therm manifestation of phototherapy treatment. 1.4 Theoretical importance and practical value of the workThe innovative nature of the work consists in addressing a current problem in elevated bilirubin levels in neonatal period, highlighting the particularities of the evolution of causes, physiopathological mechanism ,hazard and beneficial effect on the newborn , and possible complication with most accepted method of treatment. Present research, with investigation of particularities of evolution complications and revealed the need of individual approach in each case in order to reduce the neonatal risk, toward reducing neonatal morbidity and improving neonatal outcome.
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II.Literature review 2.1 Heme and bilirubin metabolismBilirubin is the potentially toxic catabolic product of heme metabolism. Heme originates in erythropoietic sources such es hemoglobin and non erythropoietic sources such as cytochromes, proxydase, and catalase. Structurally heme consists of a ring of four pyrrolees joined by carbon bridges and central iron atom structure known as ferroprotoporphyrin IX. Catalytic degradation of heme and formation of bilirubin is generated by two main enzymes, heme oxygenate and biliverdin reductase. Heme oxygenase responsible for opening of the porphyrin ring by oxidation of the alpha-carbon bridge. By that forming Biliverdin witch is green pigmented substance. Then by action of enzyme biliverdin reductase forming bilirubin IX-alpha which have orange-yellow pigment.In first chemical reaction seen on the left side of the scheme ,iron is liberated and alpha bridge carbon eliminated as carbon monoxide. The bilirubin is poorly water soluble at physiological pH due to internal hydrogen bonding that engages all polar groups.Those internal hydrogen bonding is critical in producing bilirubin toxicity and also preventing its elimination. Disruption of the hydrogen bonds is essential for elimination by the liver and the kidney.This occurs by glucuronic acid conjugation of the propionic acid side chains of bilirubin.
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2.2 Description and possible cause in elevation of unconjugated bilirubin There is 2 main categories of jaundice in neonates and can be divided into physiological and pathological causes. Physiological, appears on the 2nd-3rd day of life, with peak bilirubin levels lower than 13 mg/dL, and rate of bilirubin rise is lower than 5 mg/dL per day. Pathological jaundice may appearing the first 24 hours of life, with bilirubin rise greater than 5 mg/dL/day, bilirubin is greater than 13 mg/dL, and direct bilirubin more than 2 mg/dL at any time. The causes of hyperbilirubinemia with respect to bilirubin metabolism are numerous. In concert of red blood cell count, the normal newborn hematocrit is between 42 to 65 percent. Elevated hematocrit higher than 65 described as polycythemia, due to increase red blood cells production as can be seen in chronic hypoxia, intra uterine growth restriction, post mature infant, Infants of diabetic mother, and maternal Graves’ disease. Or, by extra red blood cells entering the circulation, as example delayed cord clamping or twin-twin transfusion. Other possible cause is increased red blood cells hemolysis, that are immune mediated with positive direct Coombs test. Usually occurs in blood groups and subtypes incompatibility. As example Rh negative mother with Rh positive baby, cause classic hemolytic disease of the newborn (erythroblastosis fetalis), ABO incompatibility witch mostly seen in type O mother and either type A or B baby, is the most common reason for hemolysis in the newborn. Other minor blood group incompatibility such as Kell witch is very antigenic (Kell negative mother) are possible but less common. Increased hemolysis which is non-immune mediated with negative Coombs test, requires blood smear to evaluated the cause. If the smear show characteristically looking RBCs, then the possible cause is membrane defect such as spherocytosis or ellipsacytosis. If the smear shows normal looking RBCs, then the possible cause are enzyme deficiency, as example G6PD deficiency , pyruvate kinase deficiency. 7

Bilirubin is then bound to albumin and carried in the blood to the liver, bilirubin may be uncoupled from albumin in the blood stream to yield free bilirubin as can be seen in neonatal sepsis, certain drugs such as ceftriaxone, hypoxia and acidosis. Bilirubin is transported to the hepatocytes, than within the hepatocytes there is conversion of unconjugated fat soluble bilirubin, to conjugated water soluble bilirubin by the action of hepatic glucuronyl transferase enzyme (GT). Decrease enzymatic activity of GT is seen in normal newborn in the first week of life, and in primary liver disease, or systemic disease affecting the liver (eg. Sepsis,TORCH, metabolic disease), or in newborns with no GT activity such as Crigler-Najjar syndrome. Then there is transport through the intahepatic biliary system for excretion into the duodenum via gallbladder and bile ducts , abnormalities of transport and excretion lead to a conjugated hyperbilirubinemia (more than 2mg/dL) as examples In case of biliary atresia. Then normally intestinal transport and excretion mechanism take a roll. Most bilirubin is eliminated in the stool with final products that are synthesized with help of colonic bacteria. Some bilirubin is eliminated in the urine, some is reprocessed in the liver due to enterohepatic circulation (along with bile acids ). Intestinal beta-glucoronidase hydrolyzes glucoronide-bilirubin bonds to yield some unconjugated bilirubin ,witch is absorbed into the portal circulation and transported back to the liver to be acted upon by hepatic glucoronyl transferase. In case of increased enterohepatic circulation such as in cases of intestinal obstruction and decreased colonic bacteria (first week of life , prolonged antibiotics and severe diarrhea) as well elevates the levels of unconjugated bilirubin, and can be a pathological cause. Breastfeeding jaundice in the first week of life also can be a possible etiology, insufficient intake of breast milk, leads to dehydration and decrease bilirubin delivery to the liver and decrease conjugation and elimination, thereby exacerbating the unconjugated hyperbilirubinemia and jaundice, therefore decrease bilirubin elimination and increased etrohepatic circulation. Those previously mentioned elevated unconjugated bilirubin, generally no requirers pharmacological treatment. 8

Adequate breast feeding (>10-20 minus per breast every 2-3 hours) will solve the underlying manifestation.
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2.3 The beneficial effect of uncongucated bilirubinemia The antioxidant effect of bilirubin was presented by several clinical studies, and shows direct correlation between concentration of BR and total plasma antioxidant capacity. Anti-inflammatory effects of bilirubin is seen as participant in physiological cascades of reaction involving the formation of acute phase reactants and some regulation of them. The immediate reaction called an acute phase reactants formation response takes place 24-48 hours after the primary insult. This first response includes initial tissue injury, resulting in a cytokine release and receptor binding with subsequent activation of signaling cascades followed by the synthesis and release of acute phase proteins. Cytokines belong to a group of small soluble peptides and they are synthesized and released from various cell types during inflammation. The pro-inflammatory cytokines include tumor necrosis factor (TNF-α) that activates endothelium causes white blood cell recruitment, and increase vascular leaks as well can mediate fever, interleukin-1 (IL-1 )witch as well participate in generation fever and activation of endothelium to express adhesion molecules, interleukin-6 (IL-6)that mediate fever and stimulates production of even more acute phase proteins, and interferon (IFN γ) witch is secreted by NK cells and T cells in response to antigen or IL-12 from macrophages and responsible to stimulates macrophages to kill phagocytksed pathogens. Secretion of these cytokines is under the control of nuclear factor kappa B (NF-κB). NF-κB is a transcription factor and regulator of numerous genes involved in the immune and inflammatory responses. In resting cells, NF-κB is located in the cytoplasm in inactive state bound to the inhibitor IκB which prevents its translocation into the nucleus. When IκB is dissociated, NF-κB is free to move to the nucleus and activates specific genes. Dissociation of IκB requires phosphorylation of this factor. Phosphorylated IκB is then ubiquitinated and selectively degraded by the proteasome complex. It was found that higher concentration of BR could suppress some pro-inflammatory cytokines (IL-2, TNF- α, IFN). It was hypothesized that BR could prevent translocation of NF-κB to the nucleus probably by inhibition of 10

phosphorylation IκB. This effect could be also induced by the heme oxygenase enzyme, that is responsible for bilirubin production. First clinical evidence of anti-inflammatory effects of bilirubin was observed in patients with rheumatoid arthritis over 75 years ago. These patients showed a remission of symptoms after developing jaundice from liver disease. Moreover, the negative association between concentration of BR and incidence of chronic inflammatory diseases has been found in individuals with Gilbert syndrome (who have chronically mildly elevated BR in serum) . Another example is systemic lupus erythematosus, an inflammatory disease associated with an increased oxidative stress and characterized by complement system aberrations, defects in antigen presentation and an abnormal adaptive immune response. Male as well as female patients with systemic lupus erythematous had almost fifty percent lower serum total BR levels than healthy controls . Those patients with the lowest concentrations of serum BR were more likely to have multi organ disease secondary to lupus. A large epidemiological study confirmed that higher total serum BR levels were associated with a reduced risk of rheumatoid arthritis in humans . However, the clear role of BR in inflammation processes has not been fully established yet. Immunomodulatory effects of bilirubin suggests that BR could possess also immunomodulatory properties. The first evidence about effects of BR on the functions of cells of the immune system was published almost thirty years ago. It was found that hyperbilirubinemia exerted a suppressive effect on antibody formation in newborn infants which supported the hypothesis that BR plays a role in the development of the immune system. During last years, it was discovered that BR significantly inhibited antigen-specific and polyclonal T cell responses, for example high levels of BR induced apoptosis in reactive CD4 T cells and inhibited cell-surface expression of MHC II class molecules on antigen-presenting cells. Another study showed a possible influence of BR on the expression of Fc receptors in macrophages. Recent investigations suggest that BR can induce expansion of regulatory T cells (Treg) which are involved in the prevention of autoimmune diseases and in the suppression 11

of the immune system tolerance probably by the induction of regulatory gene Foxp3. For a brief reminder genetic deficiency of FOXP3 can be seen in IPEX syndrome (Immune dysregulation ,Polyendocrinopathy, Enteropathy, X-linked) characterized as well by nail cystrophy, dermatitis, and/or other autoimmune derma tologic condition. It was also published that bilirubin prevents formation of atherosclerotic plaque by inhibiting monocyte migration across the vascular endothelium through disruption of vascular cell adhesion molecule-1 (VCAM-1) signaling in vitro and ameliorates VCAM-1-mediated airway inflammation in vivo.
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Adaptive response of neonatal sepsis-derived Group B Streptococcus to bilirubin The most common bacteria involved in early-onset neonatal sepsis are Streptococcus agalactiae, commonly called Group B Streptococcus (GBS).Previous studies show bilirubin has antioxidant properties and is beneficial in endotoxic shock, little thought has been given to whether bilirubin might have antibacterial properties. Analysis demonstrated 19 differentially expressed genes, almost exclusively up regulated in the presence of bilirubin. Identified 12 differentially expressed proteins, half over expressed in the presence of bilirubin. Functional analysis revealed a differential expression of genes involved in transport and carbohydrate metabolism, suggesting bilirubin may impact on substrate utilization. The data improve our understanding of the mechanisms modulating GBS survival in neonatal hyperbilirubinemia and suggest physiological jaundice may have an evolutionary role in protection against early onset neonatal sepsis. Patient distinct GBS strains were identified from septic neonates from Aberdeen Maternity Hospital, UK between January 2009 and March 2011 (the data was online published only in 24 April 2018). After several laboratory procedures including bilirubin impact on growth and GBS transcriptome, proteome analysis, RNA extraction and purification, bacterial mRNA library preparation, RNA-seq,statistical analysis of RNA-Seq data and proteomics the analysis, show that bilirubin caused a significant reduction in GBS growth ( at 24 hrs there was a 33% reduction in GBS growth p = 0.02) at concentrations above 100 µmol/l when compared to growth in the absence of bilirubin as well at this level the bilirubin switch from a potent antioxidant to a pro oxidant function. In order to elucidate the impact of bilirubin on GBS physiology they performed a whole bacterial transciptome and ptoeome analysis of GBS in the present or absence of bilirubin. Analysis of gene expression by RNA-sqe identified 19 genes that were differentially expressed at 100 µmol/l bilirubin concentration when compared with the vehicle control. The majority of genes (18/19) 13

had increased expression levels in the presence of bilirubin. Most of the over expressed genes were involved in hydrolase and transport functions. One of the most significant differentially expressed genes was over expression of sag1901 a glucuronyl hydrolase which is involved in degrading glycosaminoglycans and can effectively bind bilirubin as shown in in-vitro models and is therefore possible that GBS is increasing glucuronyl hydrolase production in response to the presence of bilirubin as an innate defense mechanism in order to bind the perceived toxin. Genes encoding transport proteins were also unregulated in the presence of bilirubin. These included sag1925 and sag1441, which both encode components of ABC transporters involved in the transport of sugars. ABC transporters are known to translocate a wide variety of endogenous and exogenous substrates across cell membranes and have previously been linked to protecting cells from bilirubin toxicity .Up regulation in the presence of bilirubin could reflect expulsion of toxic bilirubin from bacterial cells, or an increased requirement for sugar uptake from the culture medium to compensate for increased metabolic demands. Proteomic assessment of bilirubin impact show that alternation in the abundance of 22 proteins was obsessved when GBS was exposed to bilirubin compared to control. from those 22 proteins 14 of the increased while 8 were decreased. Those proteins that increased are related to transporters ,validating the gene expression and those proteins that decreased were related to regulation, synthesis or enzymatic activities. Conclusion this study was principally limited by a small number of bacterial isolates , however it demonstrates important proof of concept data supporting the hypothesis that bilirubin bay have a recognized antibacterial properties of potential importance in neonatal sepsis by common cause as GBS. Although GBS is responsible for a significant number of early-onset sepsis cases, it is not the only pathogen linked with this. Hyperbilirubinemia is a recognized feature of sepsis in neonatal, pediatric and adult populations and there is evidence to suggest that heme catabolism is increased in the critically ill patient reflect the possible mechanism if innate immune system and require further research. 14

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2.4 Witch bilirubin is neurotoxic Regional and Cell-Specific Responses to Bilirubin in the CNS The factor that have the greatest influence to treat an infant who have marked elevated bilirubin with the goal to prevent acute bilirubin toxicity is based primarily on the total serum bilirubin level. However this levels alone, is of limited value in predicting neurological impairment in newborn. The total serum bilirubin level is the level of albumin-bound bilirubin. That unbounded bilirubin levels have the strongest influence in causing neurologic injury. Unbound circulating bilirubin is in dynamic equilibrium with extravascular tissues, including the CNS, and it provides a measure of the relative amount of bilirubin that will exit the vascular space at a given level of total serum bilirubin, the albumin concentration, and the albumin–bilirubin binding constant. The latter two values have variable measurements in newborns. The bilirubin-binding capacity of albumin is reduced in infants in unstable condition, and is also reduced by the presence of competing compounds and by low serum albumin levels. Low albumin concentration have an increase the bilirubin-albumin binding affinity, however this conditions are seen only at very low albumin levels witch are not commonly seen in neonates. Even that the bilirubin have direct effect in the interaction with the brain, bilirubin level alone does not determine the risk of encephalopathy. Neurotoxicity is depends on a complex of factors such as levels and duration of exposure to high levels of bilirubin by cells of central nervous system and the innate cellular characteristics of the developing CNS that may play a roll in predisposition to damage or as protecting factor against neural injury induced by bilirubin. The threshold in witch unbounded bilirubin have a neurotoxic effect in not clear. Nor the levels in witch cellular response occurs nor levels in witch irreversible changes in tissue occurs. As a consequence there is limited data exist on the values of unbound circulating bilirubin that can be used as fixed thresholds of treatment initiation. The strongest predictors of adverse outcome by far were gestational age and birth weight, these factors may also have been related to differences in the binding affinity of albumin for bilirubin at various developmental ages. The regional 16

CNS topography and cell specific nature of bilirubin induced CNS injury is variable and there is only specific subgroups of neurons in specific areas of the basal ganglia, brain stem and cerebellum that shows great changes. This pattern is markedly different from the neuropathology features of hypoxic, ischemic brains injury during initial period of life.The region-specific nature of kernicterus could reflect differences in neurotoxic bilirubin exposure due to differences in bilirubin uptake, tissue binding, and clearance or to differential cell sensitivity to injury. CNS bilirubin uptake is passive and constant, with lipophilic unconjugated bilirubin readily permeating the tight blood– brain interfaces. Similarly, there is little evidence to suggest regional differences in bilirubin tissue binding in the CNS. Bilirubin appears to be cleared from the CNS by transporter driven efflux at the blood–brain and blood–cerebrospinal fluid barriers, cellular metabolism, or in crossover. Recognized bilirubin plasma membrane CNS efflux pumps include at least two types of transporters. ATP-binding cassette transporter B1 (ABCB1) P-glycoprotein, which is localized to the luminal (blood-side) face of capillary endothelial cells of the blood–brain barrier, and ATP-binding cassette transporter C1 (ABCC1) multidrug resistance– associated protein 1 (MRP1), which is localized to the basolateral face of the choroid plexus epithelium of the blood cerebrospinal fluid barrier. In both rodents and humans, ABCB1 and ABCC1 are the most abundantly expressed ABC transporters at their respective CNS interfaces in the developing and mature CNS. Bilirubin-metabolizing enzymes in the brain, such as cytochrome P-450 (CYP), may have a role in setting the cerebral cell-specific and region-specific toxicity of bilirubin.Oxidation of unconjugated bilirubin is catalyzed by CYP monooxygenases. A recent study showed a close inverse relationship between brain bilirubin content and expression of CYP messenger RNA, suggesting that CYP enzymes may have a role in protecting selected brain areas from bilirubin toxicity. Indeed, in studies involving the Gunn rat (a model of kernicterus), the cerebellum and the inferior colliculus, two regions that are classically affected in kernicterus, had delayed induction of CYP enzymes, as compared with induction in the cerebral cortex 17

and superior colliculus, areas that are typically unaffected. The marked difference in unconjugated bilirubin accumulation between the inferior colliculus and the superior colliculus, which are in close proximity, is unlikely to be due to differential blood supply or blood–brain barriers and is probably linked to regional differences in the cellular mechanisms for unconjugated bilirubin removal. In vitro studies have shown important neuronal and non-neuronal cell-specific responses to unconjugated bilirubin. These findings suggest that there are additional interacting and detailed mechanisms of unconjugated bilirubin toxicity.
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2.4.1 Responses of Non-Neuronal Cells Non-neuronal cells in the CNS also show sensitivity to unconjugated bilirubin, such cells include astrocytes, microglia, oligodendrocytes, brain microvascular endothelial cells of the blood– brain barrier, and the choroid plexus epithelial cells of the blood–cerebrospinal fluid barrier. The responses of these cells may play a role in modulating bilirubin-induced neurotoxicity. Primary monotypic astrocyte cultures react to toxic unconjugated bilirubin levels by secreting inflammatory mediators (interleukin-1β, tumor necrosis factor α [TNF-α], interleukin-6 through mitogen-activated protein kinase transduction, and nuclear factor κB), releasing glutamate and ultimately undergoing apoptosis. Notably, astrocytes are less sensitive than neurons to damage from unconjugated bilirubin. Similarly, microglia are directly activated by unconjugated bilirubin when placed in monotypic primary culture, assuming a phagocytic phenotype, secreting pro-inflammatory cytokines TNF-α and interleukin-1β, and showing increased activity of matrix metalloproteinases 2 and 9. Astrocytes and microglia in culture show evidence of a rapid response. Immunoreactive cytokines detected in culture medium suggest, by extension, that there is probably a strong neuro-inflammatory response during bilirubin encephalopathy. Oligodendrocytes are also susceptible to unconjugated bilirubin toxicity, with reduced mitochondrial function, increased levels of reactive oxygen species, and increased caspase 3–mediated apoptosis in the presence of unconjugated bilirubin in vitro. Studies are needed to determine whether oligodendrocyte damage impairs myelin synthesis and proper axonal function , phenomena observed in brain areas that are generally affected by kernicterus. In addition to expressing ABCB1, cultured vascular endothelial cells of the blood–brain barrier respond to hazardous levels of unconjugated bilirubin with an early increase of caveolae, caveolin-1, vascular endothelial growth factor (VEGF), and VEGF-receptor expression, followed by a 19

reduction in tight-junction protein expression; the latter suggests an adverse alteration in barrier properties. However, an alteration of the blood–brain barrier has not been observed in vivo during severe spontaneous hyperbilirubinemia. When exposed to high bilirubin concentrations, epithelial cells of the choroid plexus blood–cerebrospinal fluid interface show down-regulation of ABCC1 expression both in vitro and in vivo without an alteration in integrity of the barrier.
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2.4.2 Effect of Bilirubin on Neurons Bilirubin binds to cell membranes, especially myelin rich membranes, making neurons the principal target of bilirubin toxicity. Exposure of neurons to unconjugated bilirubin in vitro is often accompanied by macroscopic changes, including reduced dendritic and axonal arborization, reduced neurite extension and ramification, reduced cell proliferation, and increased death by apoptosis. Bilirubin delays S-phase progression and leads to cell-cycle arrest in neuroblastoma cells.This anti proliferative effect suggests that the cerebellar hypoplasia that is characteristic of murine kernicterus models may result from such cell cycle arrest. Altered cell proliferation may also adversely affect cell migration and synapse formation. Biochemical perturbations induced by bilirubin include protein oxidation, lipid peroxidation, reduced cellular glutathione content, increased lactate dehydrogenase levels, and nitric oxide release (through neuronal nitric oxide synthase activation by engagement of the N-methyl-d-aspartate receptors).Thus, bilirubin-induced oxidative stress and mitochondrial changes may be a nexus of neuronal injury. Hazardous unconjugated biliubin levels are associated in vitro with reduced oxygen consumption, cellular energy failure, reduced inner mitochondrial membrane potential, increased intracellular calcium accumulation, and activation of the mitochondrial apoptotic pathway, with caspase 3 activation and poly(adenosine diphosphate–ribose) polymerase cleavage. Moreover, N-acetylcysteine, a glutathione precursor, and glycoursodeoxycholic acid, a bile acid antioxidant, counter adverse alternations in redox status, limit oxidative stress induced by unconjugated bilirubin in vitro, and enhance cell survival. Bilirubin can also induce protective mechanisms, as shown in vitro by the marked up regulation of expression and activity for the Na+ independent cystine–glutamate exchanger system Xc(−) (SLC7A11 and SLC3A2) genes resulting in higher cystine uptake and increases in intracellular glutathione content with a consequent protection from an oxidative insult.Whether the effect is protective or toxic depends on the bilirubin concentration; this is also shown in astrocytes, where up-regulation and intracellular reallocation of the ABCC1 MRP1 21

transporter are effective at a low concentration but fail at higher concentrations of unconjugated bilirubin. 2.5 Potential long-term effects of phototherapy and associated complications.Phototherapy is commonly used to treat jaundice in newborns. It have major roll to prevent a high total serum bilirubin levels to reach a level that might be neurotoxic or require exchange transfusion. In September 24, 2018 Official journal of the American academy of pediatrics public a new research data based in recent Danish study that shows an increased risk of childhood epilepsy after phototherapy but by major difference between boys and girls. They collect 499,642 infants born after 35 weeks of gestation between 1995-2011 that was evaluated during period grater that 65 days. The total number of 37,683 infants received any phototherapy. The mean follow up time was 8.1 years. The crude outcome was 1.24 among phototherapy group and 0.76 among those unexposed. However even after adjusting for bilirubin values, the risk was statistically significant for boys (HR 1.33, 95% CI 1.1-1.61) but not for girls (HR 1.07, 95% CI 0.84-1.34).The risk of developing seizures in boys (3.7 per 1000 children) and lower in girls (0.8 per 1000 children). However, the study did not provide details on what types of phototherapy devices were used, the duration of phototherapy exposure, nor the irradiance used. Other possible outcome that was notes are Childhood cancer, melanocytes nevi, unknown retinal effects and association with childhood asthma .Concerns that neonatal phototherapy may increase the risk of cancer have been raised. However it was difficult and challenging to determine the direct association from the sat data and to control variabilities. However this concern serves as a reminder that phototherapy should be prescribed judiciously and the variables must be evaluated appropriately such as measuring the dose, use appropriate narrow light source, and use the source of light at minimum duration to minimize the risk of bilirubin induced neurotoxicity from one hand and from other hand to prevent 22

treatment association complications. The overall rates were for any cancer (adjusted hazard ratio [aHR] 1.0, 95% CI 0.7-1.6), any leukemia (aHR 1.6, 95% CI 0.8-3.5), nonlymphocytic leukemia (aHR 1.9, 95% CI 0.6-6.9), and liver cancer (aHR 1.2, 95% CI 0.2-12).
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