Introduction
Intraventricular hemorrhage (IVH) is a severe neurological disorder of preterm infants, affecting about 20 percent of the preterm infants born at or below 32 gestational weeks [
1]. Up to one half of infants with IVH develop post-hemorrhagic hydrocephalus (PHH), which is the most common etiology of pediatric hydrocephalus in North America [
2]. Despite advances in the medical and surgical care of these infants, the prognosis of preterm IVH with PHH shows unacceptable high rates of persistent neurocognitive deficits (up to 85%) and cerebral palsy (up to 70%) [
3]. Current treatment methods focus on draining cerebrospinal fluid (CSF) to reduce the excess pressure regardless of hydrocephalus etiology. There have been no substantive advances in the treatment of PHH in recent years, and few viable targeted therapeutic strategies have been proposed. To improve the care and outcomes for these patients, we must first define the fundamental mechanisms underlying the pathophysiology of IVH and its sequelae, and develop experimental models for testing therapeutic interventions.
An association between IVH, PHH, and neurodevelopmental impairment is well-established, but the mechanisms linking these disorders remain unclear. Recent evidence implicates impairment of cell junction complexes within the ventricular zone (VZ) [
4‐
10] with associated ciliopathy in the etiology of congenital, non-hemorrhagic hydrocephalus [
11‐
19] in both experimental models [
4‐
6,
10,
11,
20‐
24] and humans [
25‐
28]. VZ disruption is also identified as a key feature in IVH in humans [
29]. Therefore, an understanding of the complex effects of the blood on the VZ is needed to develop preventive treatments for neurological sequelae of IVH/PHH. This protocol describe an in vitro experimental strategy that provides the ability to control the environment of murine VZ development. The reductionist approach allows a rapid and direct method to address questions related to mechanisms and effects of blood-related VZ disruption with high temporal and physiological resolution.
The methods detailed herein are uniquely applied to IVH, PHH, or hydrocephalus more generally. While other groups have cultured ependymal cells (EC) previously [
30,
31], the notion of using VZ cultures to study the effects of IVH is a novel strategy reported from our group [
32]. This protocol is a major step forward in IVH/PHH research, as it enables rigorously controlled mechanistic experiments, with the added flexibility of mimicking various human physiological parameters. For example, studies of the VZ from patients with PHH [
29] and congenital hydrocephalus [
27,
28] indicate that alterations in adherens junction molecules play an important role in EC damage. Furthermore, the VZ culture system eliminates problems such as drug bioavailability, the blood brain barrier, and it allows high-throughput testing of the effect of drugs. Testing of pharmacological agents in vitro is an important first step to not only assess efficacy but also determine optimal ranges of dosing prior in vivo testing.
It is well known that normal multiciliated EC differentiate from monociliated stem cells; however, the complex developmental mechanisms governing this process are still not completely clear. For example, how polarity is established in multiciliated EC and how trafficking of adhesion molecules to the cell membrane occurs, requires further study. Our model will be useful for the study of normal developmental neurobiological processes, the myriad of secondary newborn brain disorders (e.g. hypoxia, inflammation, nutrition and metabolic disorders), and therapeutics explorations designed to mitigate the neurological effects of VZ injury.
The VZ is a layer of tissue mainly composed of neuro-stem cells that line the fetal ventricular system [
33,
34]. This layer is a proliferative area involved in the neurogenesis (intermediate progenitors, neurons) and gliogenesis (astrocytes, oligodendrocytes, ependymocytes) [
35] of the brain; therefore representing an important platform for brain development. Our model enables investigation of the effect of blood on these precursor cells and specifically the impact on differentiation from VZ to multiciliated EC, the final step in the transition of the VZ. EC are involved in control of fluid movement between brain extracellular space and the CSF, in part to clear metabolites from the interstitial fluid [
36]. EC also promote localized movement of fluid and its contents within the ventricles through the synchronized beating of cilia [
37]. In fact, deficits in the formation of cilia cause serious disorders in the central nervous system, including hydrocephalus (reviewed in [
38‐
40]). Despite the importance of the VZ in brain development, there are few reported methods to differentiate EC from brain neural stem cells (NSC) [
30,
41‐
43]. We have recently reported on the effects of syngeneic blood on the VZ using an in vitro model [
32], but the details of our procedures have not been published.
Discussion
Despite the high prevalence and poor outcomes of PHH, the pathogenesis of this disorder remains poorly understood [
49,
50]. One major factor limiting scientific progress in this field is a lack of appropriate models to probe the basic biology of the disease and to test novel therapeutic strategies. The current effort describes the protocol of the first in vitro model of preterm IVH/PHH and represents a major step forward for investigators. The in vitro IVH VZ model is a reliable tool to define the biological mechanisms involved in this devastating disorder.
To develop this model, an accurate isolation of the lateral wall of the lateral ventricle is a fundamental requirement. We have developed a straightforward and rapid technique to dissect the lateral ventricle by using two ultrafine forceps with just a single step to remove the thalamus, the medial wall of the lateral ventricle, and the hippocampus, versus the 5 steps previously described ([
30,
31]).
The skull dissection must be done on cooled ice (Fig.
1) to provide enough stiffness to the fresh brain tissue; otherwise, the tissue may lose its shape and attach to the frontal and parietal bones complicating the bone removal.
To obtain a cell culture that mimics the cellular composition of the VZ with fidelity it is critical to totally remove the hippocampus and the meninges to prevent proliferation of neuronal-committed cells and fibroblasts, respectively. The cells are expanded for 3–5 days or until confluence. At this time point the differentiated cells are mechanically discarded. At the time the differentiation media is added, the cultures are composes of undifferentiated NSC that correspond to newborn lateral wall VZ development reported by Delgehyr et al. [
30].
The most innovative process in this model is the addition of blood onto the cultured VZ to mimic the effects IVH on maturing neuroepithelial/ependymal cells. It is key to apply freshly obtained, whole blood onto the VZ cells as soon as possible (i.e., within 5 min). Within those 5 min, it is highly advised to keep the blood in ice cooled 25 mL falcon tubes to prevent coagulation. The presence of clots will impair pipetting the designated amount of blood onto the cells due to a pipet tip obstruction.
Likewise, this model is useful for studying the blood-related VZ cytopathology but does not take into account other physiological factors active in hydrocephalus such as increased intracranial pressure, which has been recently modeled using a 3-D neural cell cultures and a newly developed pressure controlled cell culture incubator [
51].
While other authors have shown in vitro congenital hydrocephalus-dependent cytopathology [
24,
42], this technique is a highly specific method to study the cytological mechanisms involved in the VZ after IVH. In this protocol paper, our aim is to describe our model [
32] and show representative results. Despite only showing representative results from 3 to 48 h after blood induction, this technique provides a high temporal-resolution tool that opens the possibility to study the cellular response within seconds or minutes, which is problematic in vivo. This protocol also allows investigators to employ drugs to modulate and understand normal VZ development compared to IVH conditions without in vivo impediments, such us crossing the blood brain barrier in drug delivery or tissue dissections.
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