Miltex

Five male castrated Holstein-Friesian calves 133 to 149 days old (weight range = 82–112
kg) were included in the study. The animals were sourced from a commercial high health
herd and confirmed as negative for BVDV via PCR prior to study commencement. The animals
were housed in 1 room (22m^{2 (link)}) in a high-containment (SAPO4) animal facility at the Pirbright Institute. Bedding
material was provided (https://www.mayofarmsystems.co.uk/mayo-mattress-stable-mat/), light/dark
cycle was 12:12 h, temperature was held between 10°C to 24°C, and humidity 40% to 70%.
Animals were fed concentrated rations twice daily and given ad lib access to hay and
water. Environmental enrichment was provided, including rubber toys and a hollow ball
stuffed with hay.
Four of the 5 animals (calves Nos. 2–5) were randomly assigned to the treatment group and
the remaining animal (calf No. 1) to the untreated group. The 4 treated animals were each
inoculated with 3 ml of a LSDV suspension at a concentration of 1 × 10⁶ PFU/ml;
2 ml (2 × 10⁶ PFU) was inoculated intravenously (IV) into the jugular vein, and
1 ml (1 × 10⁶ PFU) injected intradermally (ID) into 2 sites on each side of the
neck (0.25 ml in each site). The untreated animal was not inoculated. Each animal was
examined daily for clinical signs, including fever, anorexia, depression, and for gross
lesions, including cutaneous nodules and lymphadenopathy.
Skin biopsies were carried out on the 4 inoculated animals at 5, 9, 11, 15, 17, and 19
days postinoculation (DPI). Hair was removed from the biopsy site with electric clippers
and cleaned with skin wipes containing 2% chlorhexidine in 70% alcohol (Clinell, GAMA
Healthcare); 2.5 ml of lignocaine (Lidocaine Hydrochloride injection 2%, Hameln
Pharmaceuticals) was injected subcutaneously, and after 10 minutes, a 0.8 cm punch biopsy
was taken using a disposable biopsy punch (Integra Miltex). One half of the biopsy tissue
was placed into 10% sterile buffered formalin (Merck) for a minimum of 48 hours. One
quarter was placed into 4% paraformaldehyde (Santa Cruz Biotechnology, sc-281692). The
remaining quarter was stored at –80°C for future studies. Insects were fed on the skin of
the 4 inoculated cattle at up to 7 time points during the study. The results of this
procedure are reported separately (manuscript in preparation).
The 5 animals were euthanized at 19 to 21 DPI with an overdose of barbiturate solution
(Dolethal 200mg/ml Solution for injection, Vetoquinol). A postmortem examination was
carried out and tissue samples collected into 10% sterile buffered formalin for a minimum
of 48 hours before processing.

Monkeys were trained to extend a leg and remain stationary during venipuncture through a process of reinforcing successive approximations with food treats. Once trained, blood samples were taken from the lateral saphenous vein and collected in silicone coated serum vacutainer tubes for serum and K3EDTA coated vacutainer tubes (BD Biosciences, Haryana, India), for plasma. Samples were collected prior to the morning dose of the antipsychotic and therefore represented trough levels. Blood in silica coated tubes was centrifuged at 1600 x g for 30 min. Serum was collected apportioned in 0.5 ml aliquots then stored at −80°C until assayed. Blood in K₃EDTA coated tubes was centrifuged 1000–2000 x g for 10–15 min at 4°C, then apportioned in 0.5 ml aliquots and transferred to sterile polypropylene tubes. Aliquots used for blood chemistry analysis were stored at 4°C and shipped accordingly while aliquots for analysis of drug concentrations or biochemical assays were stored at −80°C until assayed.
After the 6-month treatment protocol, monkeys were sedated with ketamine followed by an overdose of sodium pentobarbital then transcardially perfused with ice cold phosphate buffered saline. Brains were removed and cut into four mm thick coronal slabs using a brain matrix (Ted Pella, Redding, CA, USA; Cat# 15039). Brain slabs were rapidly frozen on metal plates cooled by dry ice and stored at −80^oC. Time from the beginning of transcardial perfusion to freezing of brain tissue was designated as the post-mortem interval (PMI) and ranged from 38 to 87 min and did not differ significantly between groups. Brain tissue was excised using a Miltex 3.5 mm diameter short handle biopsy punch. Care was taken to maximize consistency of the punch procedure across all brains. The ventral and dorsal banks of the principal sulcus were dissected as representative of DLPFC (Area 46). Brain tissue corresponding to the rostral and intermediate EC (Area 28) was dissected, immediately rostral to the appearance of the hippocampus, located between the rhinal fissure and the amygdala, according to anatomical landmarks and stereotaxic atlas (83 ).

Coronal brain sections of the hypothalamus were sliced using a cryostat (-2.3 to -4.5 mm Bregma, using Paxison and Watson coordinates) and the ARC was extracted with a 1.5 mm disposable Miltex biopsy punch plunger (Bar Noar Ltd). Punches from each hemisphere were immersed in RNA Save (Biological Industries, Kibbutz Beit-Haemek, Israel) for RNA extraction, or frozen on dry ice for chromatin immunoprecipitation.