Until recently, the cerebellum and its vulnerability to perinatal HI have been underappreciated and unexplored. This neglect, in part, was due to the long-held belief the cerebellum functioned only in motor coordination and did not make important contributions to non-motor, cognitive and emotional domains. Using a model of term HI in the rat, we report a diffuse injury to the cerebellum seven days post-injury, with a focused impact on Purkinje neurons that differs in males versus females. When compared to the same-sex control group, male Purkinje neurons had fewer dendrites after HI while female Purkinje neurons tended to have more. To understand the origins of these Purkinje neuron alterations, we explored a targeted list of genes associated with inflammation, steroid hormones, autacoids, neurogenesis, synaptogenesis, and phagocytosis. We found a small number of genes altered in response to HI, with very little overlap between males and females. Western blot quantification of proteins to survey gross abnormalities in different cerebellar cell types and their neurotransmitters revealed decreased glutamic acid decarboxylase (GAD) expression, suggesting that a loss of inhibitory regulation may be the source of damage to Purkinje neurons. Immunohistochemistry and volumetric analyses found no evidence of gross changes to microglia, astrocytes, granular neurons, or the number of Purkinje neurons.
Findings from a line of research, initiated a decade ago, determined a latent sensitive period in cerebellar development during the second postnatal week in the rat 23–25. This sensitive period in rats is analogous to full-term birth in humans, a time when males experience greater vulnerability to HI than females 4–7, 26. During this time endogenous prostaglandin E2 (PGE2), a lipid best known to mediate inflammation, is elevated in both sexes 23,25. If PGE2 levels are thrown out of balance (i.e., increased or decreased) by inflammation or injury, the growth of Purkinje dendritic trees is altered, and later social cognitive and social behaviors are impaired in males only 24,25. Interestingly, mRNA analysis, while not comprehensive, found evidence for facilitation of the prostaglandin synthesis pathway after HI in females. Expression of fatty acid amide hydrolase (Faah), which is the enzyme that produces the most important precursor in prostaglandin synthesis, and prostaglandin receptor subtype EP1 (Ptger1) were upregulated after HI in females only. This is intriguing given that PGE2 stunts the development of Purkinje neuron dendritic tree arborization, and EP1 activation is generally considered a source of neuronal damage following HI in cortical brain regions 25,27. We did not measure either PGE2 or EP1 protein in this study, but this finding nevertheless highlights the central role of prostaglandin signaling in cerebellar development.
In the term infant, HI is typically associated with diffuse tissue loss, or hypoplasia, and damage to deep gray matter regions including the basal ganglia and thalamus 28. Until very recently, perinatal HI was mainly characterized as a cerebral injury in the clinical setting. Nevertheless, the injury most often identified by MRI in the cerebellum of term infants experiencing HI is a bilateral, symmetrical decrease in hemisphere volume 29. Though effects of perinatal HI on the cerebellum have been observed in experimental models, results are difficult to interpret due to inconsistencies in the timing of injury. The Rice-Vannucci method (i.e., permanent unilateral carotid ligation + period of systemic hypoxia) is by far the most widely used animal model to replicate HI. Initially developed in adult rats, the model has since been adapted for use in perinatal rodents, with PN7 pups being the current standard. Studies inducing HI in PN7 pups, which is best suited as a model of preterm neonatal injury, report reductions in the number of Purkinje neurons in both the right and left cerebellar hemispheres and abnormalities in Purkinje and granule cell dendrites 30. It is currently considered that the PN10 Rice-Vannucci method, used here, is a more appropriate model for human infants at term 31. To determine the effects of term-equivalent HI on Purkinje neuron morphological characteristics we used an AAV to sparsely label individual Purkinje neurons throughout the cerebellar vermis. Purkinje cell dendritic morphology was reconstructed in 3D and revealed that HI in the neonatal rat disrupts the dendritic branching of Purkinje neurons in the cerebellar vermis. Moreover, we find that the effect of HI on Purkinje neurons differs between the sexes, with males displaying a reduction in dendritic arborization and females displaying modest but significant dendritic outgrowth. These differences suggest either that repair mechanisms are initiated earlier in females or that females are protected from HI. Thus, optimal windows for intervention might differ between the sexes. Considering the timing of HI injury, our results in the cerebellum are consistent with those reported in studies modeling preterm neonatal HI injury. Cerebellar development is delayed relative to the rest of the central nervous system and undergoes significant cytoarchitectural changes after birth 19,21. The second postnatal week in the rat cerebellum, which corresponds to birth in the human, is the most dynamic period of Purkinje neuron dendritic remodeling and a developmental window sensitive to extrinsic factors 32. During this period, the Purkinje neuron dendritic tree is rapidly expanding and organizing. The Purkinje neuron dendrites, which develop from multiple perisomatic processes, are pruned, leaving only a single primary dendrite that is innervated by a single climbing fiber. Simultaneously, Purkinje dendrites undergo tremendous growth and are remodeled from a multiplanar to monoplanar arrangement 21.
The source of the increased vulnerability of males to deleterious consequences of HI on Purkinje neuron development is unknown. Purkinje neurons, the principal cells of the cerebellum and the only efferent of the cerebellar cortex, undergo tremendous growth during the perinatal period, a time when developing males experience elevated androgens 33,34. Purkinje neuron dendritic branching and elongation extends into postnatal life, terminating with the completion of granule neuron migration and parallel fiber maturation (second to third postnatal week in the rodent and up to two years in the human) 21. Earlier animal studies have shown that among the cell types of the cerebellum, Purkinje neurons are especially vulnerable to hypoxic insults, which is most likely a result of their high metabolic rate and associated oxygen demand 35,36.
The calcium-binding protein Calbindin D28k is expressed by many types of neurons throughout the brain but is disproportionately abundant in Purkinje neurons, along with discrete populations of interneurons in the cerebral cortex and hippocampus, and so it is predominately used as a marker to identify these cell types 37. Calbindin serves a threefold function as a Ca2+ buffer, transporter, and sensor 38. While the precise function and regional specificity of Calbindin in Purkinje neurons are not fully elucidated, high expression is likely due to unique electrophysiological properties of Purkinje neurons, such as their high firing rate under resting conditions and characteristic complex spikes 35,39. Androgens developmentally regulate calbindin as well as several calcium binding proteins and cation transporters, which can increase vulnerability to excitotoxic cell death 40–42. We observed increased levels of calbindin in male Purkinje neurons, with and without HI, suggesting this may be a source of increased sensitivity to altered excitability, thereby increasing male vulnerability.
Cumulative evidence supports a link between abnormal cerebellar development and major neurodevelopmental disorders that present with a strong gender bias, including autism spectrum disorders (ASDs), attention deficit hyperactivity disorder, and developmental dyslexia 43,44. For example, perinatal cerebellum injury is the leading risk for ASDs, which are diagnosed more frequently in males than females. The two most consistent cerebellar abnormalities found in people with ASDs are Purkinje neuron loss (determined by postmortem histology) and reduced cerebellar volume (determined by MRI) 45. The latter is believed to result from reductions in Purkinje neuron number and size 46. Although developmental damage to the cerebellum, particularly Purkinje neurons, is strongly associated with neurodevelopmental disorders, the underlying pathogenesis and resultant behavioral defects, remain poorly understood. Purkinje neurons are the central components of all cerebellar circuits, the first neurons to populate the cerebellar cortex, and based on their ability to regulate the development of both inhibitory and excitatory neurons, they are considered key modulators of cerebellum development 47,48. Lurcher mutant mice, which are characterized by postnatal degeneration of cerebellar Purkinje neurons, display ataxia, deterioration of cognitive functions, increased activity, and increased repetitive behaviors 49,50. In rats, disruption of Purkinje neuron development by inflammation during the second postnatal week leads to stunting of Purkinje neuron dendrites and impairs juvenile social behavior only in males 25. Early damage to the cerebellum can have broad, enduring consequences on the developmental trajectory of the cerebellum and behavior, but further research is needed to understand this complex relationship.
It remains unclear what role microglia are playing in the progression of cerebellum injury following perinatal HI. Microglia are key contributors to neurodevelopment and mediate distinct neuroimmune responses in the healthy and injured brain. Although much of our knowledge derives from studies in the cerebrum, microglia function in neurodevelopment by promoting cell genesis, controlling cell numbers, synaptic pruning, and apoptosis 51–54. In the developing cerebellum, microglia promote apoptosis of immature Purkinje neurons and subsequently clear debris by phagocytosis 55. In perinatal brain injury, one of the major pathogenic factors is microglia-mediated neuroinflammation, and most studies associate microglia activation detected at initial stages of injury with exacerbation of brain injury 56–59. Using immunohistochemical methods we detected no changes in microglia numbers or morphology in the cerebellum one week after HI, which is likely a result of the time between injury and assessment. Interestingly, our gene expression results hint at a role for microglia in the progression of cerebellar injury after HI, given that several genes associated with microglia activation were increased in males after HI, whereas in females there was an increase in Sirpa, a complement protein known to prevent phagocytosis of stressed cells 60.
There are limitations to the current study. First is the intrinsic variability of the widely used Rice-Vannucci model of term HI. Procedural times and duration of isoflurane exposure, which we standardized and kept to a minimum, are believed to cause inconsistencies in brain injury 61,62. Second, we did not investigate the behavioral outcomes of perinatal HI injury in adult rodents. Although this model gives rise to well-documented behavioral phenotypes, including impaired spatial learning and memory, impaired motor control, and cognitive and sensory processing deficits, it would be impossible to ascertain what components of these behaviors rely on the cerebellum as opposed to the more frankly damaged cerebral cortex and hippocampus. These limitations highlight the need for a focal, term-equivalent HI model targeting the cerebellum.
Collectively, the present studies add to a growing body of literature demonstrating that HI insult causes diffuse, bilateral injury in the term-equivalent cerebellum and that Purkinje neurons are particularly susceptible. To our knowledge, this is the first report of HI effects on Purkinje neuron dendritic branching patterns. A remaining question is the mechanism underlying the effects of HI on Purkinje neuron arborization. The timing of injury coincides with the sensitive period for dendritic remodeling of Purkinje cells, which is modulated by excitatory afferent fibers, trophic factors, and hormones. We also report a reduction in GAD signaling in the cerebellar cortex following HI. This, along with the spatio-temporal location of injury, suggests that synaptic inputs to Purkinje neurons may be a target of HI. Further research is clearly needed to explore this and better understand how sex modulates HI in the term-equivalent cerebellum.