13 male Sprague-Dawley (SD) rats (Taconic Bioscience Inc., Denmark) with a imply weight of 350 g have been used. The rats have been housed in teams of 4 with entry to advert libitum customary rodent eating regimen and water in animal services (Glostrup Analysis Institute, Rigshospitalet) with managed temperature, humidity and an inverted 12-hour mild/darkish cycle. The research was accepted by the Institutional Animal Care and Use Committee (Glostrup Analysis Institute) and achieved in compliance with Danish regulation. License (2012-15-2934-00283) granted by the Danish Animal Experiments Inspectorate. All strategies and knowledge are reported based on ARRIVE pointers.
Invasive procedures and eye exams have been carried out beneath anesthesia induced with 2.7mL/kg (maintained with 1.0mL/kg each 30 min) of balanced anesthetic combination (1.25 mg/mL Midazolam (Hameln Prescribed drugs) and “Hypnorm” with 2.5 mg/mL fluanisone and 0.079 mg/mL fentanylcitrate (Skanderborg Farmacy) injected subcutaneously (s.c.).
On the day of surgical procedure and the next 2 days, anti-inflammatory (5 mg/kg carprofen, Rimadyl®, Pfizer), antibiotic (10 mg/kg enrofloxacin, Baytril®, Bayer) and analgesic (0.03 mg/kg buprenorphine, Temgesic®, RB Prescribed drugs) therapy was injected s.c. For euthanasia an intraperitoneal injection of a pentobarbital-lidocaine combination (65 µL/kg) was used.
Rats have been randomly assigned into 2 teams. Eight animals within the hydrocephalus group have been injected with a suspension of kaolin into the cisterna magna and 5 animals within the management group was equally injected with an equal quantity of the automobile (Ringer’s lactate answer). Optic disc picture (ODP) was obtained preoperatively at day 0 and once more after 7 days to permit detection of in vivo adjustments.
Epidural ICP was recorded at day 0 postoperatively and constantly till day 1 for roughly 18–22 h after which once more at day 7. In all animals the ultimate epidural ICP recording was additionally adopted by an intraventricular ICP recording to validate the epidural ICP. The intraventricular recording was carried out through the prepositioned information probe put in epicranially at day 0 and subsequently the dura and ventricle weren’t penetrated till day 7. Puncturing the dura and canulating the ventricles have been intentionally prevented initially at day 0 to keep away from neuro-infection and CSF shunting. In the long run of the research, animals have been sacrificed with a pentobarbital-lidocaine combination and the brains have been eliminated instantly and glued in a single day in paraformaldehyde (4%) in phosphate buffered saline (PBS) at 4 °C.
Induction of hydrocephalus
The anesthetized rat was positioned on a heating pad and the pinnacle was mounted in a stereotactic body (David Kopf Devices). With the neck flexed in 90 levels angle the rhomboid atlanto-occipital membrane above the cisterna magna was marked on the pores and skin between the cranium and the primary cervical spinosus. An insulin syringe with a 30 G needle was crammed with both 90 µL of a 25% sterile kaolin suspension (in automobile) or 90 µL of the automobile alone. Ringer’s lactate answer (1.4 mM Ca2+, 4 mM Okay+, 130 mM Na+, 109 mM Cl−, 28 mM lactate) was used as automobile. The needle was positioned within the beforehand marked space and slowly superior in a vertical route till there was a lack of resistance. The percutaneous injection was carried out step by step over a interval of 10 s. Following the injection, the neck was prolonged, the higher jaw mounted within the stereotactic body and the rat was noticed for respiratory melancholy.
Implantation of epidural probe and ventricular information probe
Surgical procedures and ICP recordings was carried out just like the tactic described intimately by Uldall et al. . The strategy is described in short beneath.
Native anesthesia and vasoconstrictor (10 mg/mL lidocaine and 5 µg/mL) have been injected subcutaneous within the midline of the scalp. A 2 cm midline incision was made with a scalpel from simply above the eyes to between the ears. The underlying fascia was separated with forceps and a wound retractor was inserted. The remaining fascia and comfortable tissue have been eliminated till bregma, lambda and the frontal and partial bones have been uncovered. Fastidiously, to keep away from any injury or penetration of the dura mater, 5 holes have been made with a dental drill; 1 for the epidural probe (C313G-3UP, PlasticsOne®), 1 for the ventricular information probe (C313G-0-0.4, PlasticsOne®) and three for anchoring screws. The probes have been anchored to the anchoring screws with light-curing dental resin cement (Panavia™ SA Cement Plus). The epidural probe was protected by a cap (303DCFT-1-GFN, PlasticsOne®) sealed with dental rubber cement. The ventricular information probe was protected by a cap (C313DC-GFN-SP, PlasticsOne®) with an inside metallic dummy cannula reduce 1 mm beneath the pedestal of the probe, additionally sealed with dental rubber cement. The incision of the pores and skin was closed with inverted sutures (4−0 Vicryl™ Plus, Ethicon) and lined with vaseline.
Previous to epidural ICP recordings the epidural probe was cleaned and flushed with an inside cannula (C311IU reduce in stage with the pedestal of C313G-3UP, PlasticsOne®) earlier than the tube was linked to the epidural probe.
For the only intraventricular ICP recording the interior cannula (C311I reduce 5 mm beneath the pedestal of C313G-0-0.4, PlasticsOne®) was slowly superior within the ventricular information probe till a pulsating drop of CSF was noticed on prime. The tube was then linked to the interior cannula and held in place with an outer tubing having a captive collar (C313C, PlasticsOne®).
Correct ICP hint was confirmed by performing a bilateral jugular vein compression, which transiently raised the ICP. ICP knowledge from day 0, 1 and seven was collected as a 5-minute imply preceded by 10 min of secure ICP studying. Steady ICP knowledge was collected from every animal as a 5-minute imply for each 30 min of recording.
Optic disc images
ODP was obtained from each the hydrocephalus group and the management group preoperatively at day 0 and after 7 days. ODPs have been captured with a digital fundus microscopy system (Phoenix Micron IV In Vivo Imaging Microscope, StreamPix v5.19, Phoenix Analysis Labs) designed for retinal imaging of laboratory rodents.
Rats have been anesthetized and instantly after administered pupil dilating drops (1 drop/eye tropicamide 0.5%) and positioned on an adjustable stage. Pupil dilation was decided by directing the sunshine from the picture microscope in direction of the eyes. Coupling gel (1–2 drops/eye GoniSoft ®) was utilized to guard and moist the cornea whereas the lens was superior to the attention. The optic disc and peripapillary retina have been noticed, and an photographs sequence was recorded for 10–15 s with totally different mild depth and focus level for each eyes.
Fundus recordings have been evaluated in Micron Uncover for Phoenix Micron IV In Vivo Imaging Microscope v1.2 and the best high quality ODPs have been saved as bitmap picture recordsdata.
Basic indicators of papilledema have been outlined as a number of of following: adjustments to the optic disc margin, peripapillary halo, vessel engorgement and/or torsion and/or obscurations, hemorrhages, peripapillary choroidal folds. These papilledema indicators have been used as foundation for a blinded analysis by an skilled neuroophthalmologist (SH). Day 0 ODPs and day 7 ODPs from every particular person animal have been in contrast and both marked as “no optic disc adjustments” or “optic disc adjustments”. Animals with unilateral or bilateral adjustments have been each labeled as having “optic disc adjustments”. For the animals assessed with “optic disc adjustments” the adjustments included paleness within the temporal higher quadrant of the optic disc margin and the peripapillary space.
Kaolin induced OHC might be detected anatomically with considerably dilated lateral ventricles at day 7 after profitable kaolin injection [5, 12]. To verify the illness mannequin, gross anatomical adjustments was assessed by reducing the brains in 1 mm coronal slices and midbrain slices was photographed to substantiate ventricle adjustments.
Pattern sizes have been calculated with a statistical energy evaluation (alpha = 0.05, energy 0.80) and enrolled 2:1 primarily based on a pilot research the place 25% have been sacrificed due to opposed results on hydrocephalus induction. All ICP values are offered as imply ± customary deviation (SD) if not acknowledged in any other case. ICP knowledge have been collected from Perisoft for Home windows v2.5.5 and visualized in GraphPad Prism 9 utilizing two-way ANOVA for group comparability. Linear regression evaluation was carried out in GraphPad Prism 9 and regular distribution was ensured utilizing Shapiro-Wilk check. The Banard’s precise check was carried out for group comparability of optic disc adjustments in Rstudio v4.1.0 utilizing the Barnard-package v1.8. p-values beneath 0.05 was thought of important.