Germinal matrix hemorrhage model and induction of post-hemorrhagic hydrocephalus in neonatal mice
We developed a novel neonatal mouse GMH model with relevance to human clinical disease in several aspects, including pathophysiology of insult, post-hemorrhagic survival rate, temporal profile of pathology, rate of post-hemorrhagic hydrocephalus, and motor/cognitive delay and deficits. Surgical details of the model are in materials and methods, and a workflow of the surgery and our subsequent complement inhibitor treatment paradigm is presented in figure 1. Briefly, at post-natal day 4 (P4), mouse pups were removed from their mothers and anesthetized on ice after defining syringe insertion location. Pups were then injected with 0.5 µl collagenase directly into the subventricular zone (SVZ) as annotated in figure 2a. After 1 hour on a heat pad, pups were returned to their mother. This procedure resulted in a lesion and deposition of blood products in a periventricular pattern, as shown in Nissl stains of brain sections collected 24 hours after injury (i.e. P5) (Fig. 2a). Of note, blood products were not only deposited in the ipsilateral hemisphere of needle insertion but were also evident along the ependymal layer of the contralateral ventricle (shown with a black arrow in Fig. 2a), indicating intraventricular hemorrhage, a feature of human GMH. The lesion and blood product deposition were a result of the collagenase injection and not mechanical insertion of the needle, as brain samples collected from PBS-injected animals (designated as sham) presented no lesion, blood deposition, or enlargement of the ventricles (hydrocephalus), in contrast to brains from collagenase-injected animals (Fig. 2b). In addition, unlike collagenase-treated animals, neither sham animals nor naïve non-injured pups showed any denudation of the ependymal lining in the lower border of the ventricles, shown in high resolution Nissl-stained images (Fig. 2b). The decision to induce injury at P4 was based on initial studies in which we found that collagenase treatment on P2 or P3 resulted in unacceptably high mortality rates (Fig. 2c). A grading system from 0 to 5 was developed to characterize the severity phenotypes of brain injury and hydrocephalus, with scale 5 corresponding to global hydrocephalus. Details of the scoring system are described in the methods section, and representative images corresponding to injury scale are shown (Fig. 2d). Using Ter-119 red blood cell (RBC) stain, we show hematoma along the edges of the lesion three days after injury (Fig. 2e). Of note, the central portions of the hematoma get washed away as an artifact of the staining process, but the RBC stain shows layering of RBC’s along the border of the lesion. Complement deposition was evident in the perilesional hemisphere with diffuse deposition along the lesion border as depicted by immunofluorescence staining for C3 (Fig. 2f), thus providing justification to explore the role of complement in the context of GMH and its post-hemorrhagic sequalae.
Targeting and tissue distribution of CR2Crry in neonates after induction of GMH
The complement inhibitor CR2Crry has been shown to bind deposited C3 activation fragments ([20]), which occurred at sites of injury in brains of collagenase injected mice (see above, Fig. 2e). To examine targeting specificity and whole-body distribution after systemic administration of CR2Crry, we administered fluorescently labeled CR2Crry via i.p. injection after induction of GMH. Live animal fluorescence tomography showed an initial systemic distribution of CR2Crry, with subsequent localization of the drug to the brains of GMH mice, but not to brains of control animals with no GMH (Fig. 3). Furthermore, quantification of fluorescence intensity revealed a CR2Crry tissue half-life of about 3 days in the brains of GMH mice, which we used as the interval between CR2Crry treatments in the therapeutic paradigm below.
Complement inhibition reduces lesion size and hydrocephalus in injured pups
To investigate the role of complement in GMH-induced pathology in a clinically relevant setting, we treated pups with CR2Crry or vehicle starting at either 1 or 24 hours after collagenase injection, and every 3 days (the tissue half-life) thereafter until sacrifice at P14 (refer to Fig. 1). Nissl stains of mid-hippocampal and ventricular regions from the vehicle group collected at P14 demonstrated varying degrees of parenchymal lesion along with high rates of associated intraventricular hemorrhage and PHH; no brains from the vehicle-treated group scored scale 0. On the other hand, 28% of 1-hour CR2Crry-treated animals were scale 0, and 17% of 24-hour CR2Crry-treated animals were scale 0. Global hydrocephalus occurred in 61% of brains from vehicle treated animals (scale 5), compared to only 7% and 22% in the CR2Crry 1-hour and 24-hour treatment groups, respectively (Fig. 4a and b). At P45 (41 days after injury), PHH was 75% in vehicle and 33% in the CR2Crry group (P<0.05) (Fig. 4f).
The lesion and ventricular volumes of the experimental groups were quantified using serial Nissl stained sections through each brain. Both ventricle and lesion volumes were decreased with CR2Crry treatment (both 1-hour and 24-hour treatments) compared to vehicle-treated animals (Fig. 4c and d). There was no significant difference between the 1 and 24-hour CR2Crry treatment groups, and in subsequent experiments we focused on 1-hour CR2Crry treatment. Of note, brains from vehicle-treated animals were more likely to possess bilaterally enlarged ventricles occupying the majority of the intracranial compartment, coupled with relatively large lesions, as shown in the representative 3D reconstructed images of ventricle and lesion volume of all three conditions (Fig. 4e). The lateral ventricles in brains from CR2Crry-treated animals were closer to normal ventricular anatomy compared with the visually effaced ventricles observed in the vehicle group.
CR2Crry treatment decreases perilesional complement deposition, astrocytosis, and microgliosis
We investigated the impact of complement inhibition on a perilesional cellular response in terms of post-hemorrhage astrocyte and microglia/macrophage recruitment. For analysis of astrocytosis, brain sections from P14 mice were stained for Glial Fibrillary Acidic Protein (GFAP). Astrocytosis was examined in terms of the extent of astrocytic scar extending from the lesion border inward towards intact parenchymal tissue (Fig. 5a), and in terms of astrocyte density in the perilesional area at the interface with lesion (Fig. 5b). Compared to vehicle-treated animals, CR2Crry treated animals displayed reduced ipsilateral astrocytic scar formation within surrounding brain parenchyma (Fig. 5a, b). In addition, contralateral periventricular astrocytosis was also higher in vehicle animals compared to CR2Crry treated animals (Fig. 5c). Similarly, Iba-1 staining for microglia/macrophage in the perilesional region showed reduced microgliosis in CR2Crry-treated animals compared to vehicle treated animals (Fig. 5d). Correlating with reduced astrocytosis and microgliosis, there was also reduced C3 deposition in the perilesional area (Fig. 5e) and ipsilateral hippocampus (Fig. 5f) of CR2Crry treated mice.
Complement inhibition results in dendritic and neuronal preservation
To explore the role of complement activation in the neurodegenerative process occurring post-hemorrhagic injury, we investigated the effect of complement inhibition on dendritic arborization (MAP2 stain) in ipsilateral and contralateral cortical hemispheres (Fig. 6a). Compared to naïve mice, vehicle-treated animals displayed a decrease in dendritic arborization in both ipsi- and contralateral hemispheres, which was largely reversed with CR2Crry treatment; there was not a significant difference in MAP2 staining intensity between naïve and CR2Crry treated mice, suggesting a role for complement in dendritic loss post-injury. To further interrogate perilesional neurodegeneration, we immune-stained for neurons (NeuN). NeuN signal intensity, measured in terms of distance from lesion, was markedly higher in CR2Crry-treated mice compared to vehicle controls (Fig. 6b, upper panel). Brains from CR2Crry-treated mice showed high neuronal density in the immediate perilesional space compared to effacement of perilesional neurons in vehicle treated mice (Fig. 6b, lower panel). Notably, in vehicle-treated animals there is a presence of non-neuronal cells in the vicinity of the lesion as indicated by DAPI staining.
Neuronal loss is promoted by microglial/macrophage engulfment of complement opsonized neurons.
We next investigated a role for microglia in complement-mediated neuroinflammation that is associated with loss of neuronal density. In figure 5b, we analyzed perilesional neuronal density spatially extending from the lesion. Here, we analyzed overall neuronal density within perilesional fields and show that compared to naïve animals, vehicle-treated animals had a reduction in neuronal density in the perilesional area of microgliosis that colocalizes with C3 deposition. Compared to vehicle treatment, CR2Crry treatment reduced C3 deposition and microgliosis and preserved neuronal density (Fig. 7a-b). To investigate whether C3 opsonization may be responsible for microglial association with neurons and subsequent neuronal loss by promoting microglia-dependent engulfment, we first quantified colocalization of microglia/macrophages with C3-tagged neurons. Within perilesional fields of view, C3/Iba-1 colocalization was observed on 62% of NeuN+ stained cells in vehicle-treated animals compared to 20% in CR2Crry-treated animals (Fig. 7c). We next demonstrated C3 deposition at the microglial/macrophage interface with neurons and quantified microglia/macrophage internalization of C3 and of neuronal (NeuN+) material. We found a higher number of microglia/macrophages with partially or fully internalized C3 in vehicle-treated animals compared to CR2Crry-treated animals (Fig. 7d). From calculations using the total number of NeuN+ cells within each field as the denominator, we similarly found a higher percentage of microglia/macrophages with partially or fully internalized NeuN+ material in vehicle-treated animals compared to CR2Crry-treated animals (Fig. 7e). Two examples of microglia surface interaction with and internalization of a C3-tagged neuron are shown in figure 7f. Example 1 shows a C3 tagged neuron engulfed within a microglia/macrophage, and example 2 shows a direct interaction between a C3 tagged neuron and a microglia/macrophage (see supplementary material for video demonstration). These data indicate a role for complement-dependent microglial phagocytosis in neuronal loss after GMH.
Complement inhibition improves overall weight gain and animal survival
Weight gain was monitored from P2 until sacrifice at P14. Compared to vehicle-treated mice, the overall weight gain in this period was significantly improved for mice treated with CR2Crry and was similar to percent weight gain in naïve mice (Fig. 8a and b). In the two days prior to injury, all groups were growing at a comparable percent weight gain. 24 hours after GMH induction (shown by purple arrow, Fig. 8b), there was a deceleration in percent daily weight gain in both CR2Crry and vehicle animals until 4 days after injury (P8, shown by orange arrow). At that time point, CR2Crry animals began to exhibit an accelerated weight gain and approached the normal weight gain curve, as displayed by naïve animals. There was no difference between naïve and CR2Crry animal percent weight gains by P14. In a separate cohort of animals, survival was monitored for up to 41 days after collagenase-induced injury (P45). For this experiment, the same treatment paradigm used in the above studies was applied through P14, with subsequent CR2Crry or vehicle (PBS) treatments given weekly. Animal survival assessment began one day after injury (P5) to eliminate surgery-related deaths occurring within 24 hours. CR2Crry group mortality plateaued at P25, while vehicle animal mortality continued to increase through P45, at which time survival rate of CR2Crry-treated animals was 75% compared to 40% for vehicle treated animals (Fig. 8c). Within the vehicle cohort, 4 of 10 females and 4 of 9 males survived to P45 with no significant difference in gender. In the CR2Crry group, 4 of 5 females and 5 of 6 males survived, with no significant difference in gender.
Complement inhibition after germinal matrix hemorrhage enhances motor and cognitive performance at adolescence.
An ongoing neuroinflammatory response has been linked to motor and cognitive dysfunction that is likely secondary to a loss of neurons. Our data above show an ongoing complement-dependent neuroinflammatory response and loss of neurons after GMH, and we therefore assessed whether this was linked to motor and cognitive performance at P30, when mice are able to physically perform behavioral tasks. Gait analysis (Noldus CatWalk XT) was performed at P30, and a CCI was computed using 100 plus different obtained values. Naïve and CR2Crry-treated mice had similar CCI scores, and their scores were significantly higher than vehicle-treated mice (Fig. 9a). Hippocampal integrity was assessed with fear-conditioned memory retention using the passive avoidance task. CR2Crry-treated mice showed similar retention memory to naïve mice represented by a delayed time to enter the shock box of the task, which was significantly lower in vehicle-treated animals (Fig. 9b). The Barnes maze task was used to assess spatial learning and memory retention, and as with above tasks, CR2Crry-treated and naïve mice performed similarly and significantly better than vehicle-treated mice. CR2Crry-treated mice exhibited improved spatial learning ability throughout the learning phase of the task compared to vehicle-treated mice as shown by an improved total latency on the platform and latency until first peek into the escape hole (Fig. 9c). Additionally, for both latency parameters, CR2Crry treatment significantly improved animal retention memory compared to vehicle on the final day, in which animals performed the task after a 2-day break period. Heat maps depicting the movement of animals on the platform from representative experiments are shown in figure 9d. Thus, neuroinflammation and neuronal loss after GMH correlates with behavioral deficits as mice age, and these outcomes can be reversed by complement inhibition.