C3a-C3a receptor signalling stimulates post-ischaemic neural plasticity and intranasal treatment with C3a receptor agonists is an attractive approach to improve functional recovery after ischaemic brain injury.
Stroke is the primary cause of disability in adults and the second most common cause of death. Loss of function after stroke is due to cell death in the infarcted tissue and cell dysfunction in surrounding and remote brain areas that are connected to the damaged
A study conducted on mices by an international group of scientists, published on Brain, explains how C3a protein, through nasal drops, will streamline the patient’s functional recovery affected by stroke, amplifying the plasticity of neural circuits.
Twenty-one days after ischaemia induction, mice were deeply anaesthetized with thiopental (Hospira) and transcardially perfused with 0.9% saline, followed by 4% paraformaldehyde in 0.1 M PBS. Brains were removed and immersed in the same fixative overnight. Tissue was dehydrated, embedded in paraffin, and cut into 6-mm serial coronal sections. Every 20th section was stained with haematoxylin and eosin. Infarct size was evaluated morphometrically on digital images with ImageJ software (NIH, v. 1.47q) by manual delineation of the infarct and hemisphere areas on sections spanning the entire lesion along the anterior-posterior axis by an investigator blinded to experimental group. Volume of injury was derived by multiplying area of total tissue loss that includes shrinkage due to scarring [(contralesional hemisphere ipsilesional hemisphere) + infarcted tissue] on each section by the total intersection distance.
Purified human C3a peptide (Complement Technologies) was diluted in sterile phosphate-buffered saline (PBS) to a concentration of 200 nM and a total of 20 ml (10 ml/nostril; corresponding to 1.13 mg/kg body weight) of peptide solution or PBS was given intranasally to awake, hand-restrained mice held in a supine position. Solutions were administered through a pipette tip, drop-wise in 5-ml portions divided by 1-min intervals to allow for absorption. C3a or PBS was given every 24 h on Days 7 to 21 post-stroke for the short-term study or on Days 7 to 28 post-stroke for the long-term study. Mice were assigned to C3a or PBS treatment using randomization strati- fied by body weight to avoid potential confounding effects of body weight on behavioural performance. The investigators carrying out behavioural studies and analysing data were blinded to treatment group. For the assessment of potential systemic anaphylactic response due to intranasal C3a inoculation, body temperature was monitored using a rectal temperature probe (Harvard Apparatus) inserted 4 mm into the rectum of awake mice restrained by the scruff. Baseline temperature was taken before intranasal administration and 5, 15, 30, 45 and 75 min after C3a or PBS administration.
Photothrombotic stroke was induced in the left cortex at the border between primary motor and primary somatosensory cortical areas corresponding to the forelimb. Intranasal C3a treatment had no effect on infarct volume (P = 0.429, Fig. 4D). In the grid walking task, C3a-treated mice showed a tendency toward reduced number of right paw foot faults on Days 14 and 21 compared with Day 7 (P = 0.0545 and P = 0.0839 for Days 14 and 21, respectively, Dunnett’s test), whereas no trend toward significant improvement was observed in PBS-treated mice (P = 0.147 and P = 0.486 on Days 14 and 21, respectively, Fig. 4E). At all time points after stroke, both groups showed significant impairment with respect to the baseline performance (P 5 0.001). In the cylinder test, C3a-treated mice showed continuous improvement between Days 7 and 21 such that on Day 21 their frequency of right paw use for body support did not differ from baseline performance (P = 0.062, Fig. 4F). The PBS-treated mice showed sustained impairment until the end of the testing period (P = 0.001 Day 21 versus baseline; n = 10 mice/group). These results show that intranasal C3a treatment can promote the recovery of forepaw function after ischaemic stroke.
Second Row, from left: Scott R. Barnum, Rick A. Wetsel, Jonas A. Nilsson, Mike Dragunow and Marcela Pekna.