Mice and experimental design
Experimental arthritis was induced in 7-8 week old DBA/1J male mice
(ENVIGO, Blackthorn,UK). The mice were maintained in a conventional
animal housing facility at Sheba Medical Center and kept in individually
ventilated cages. All experiments were approved and executed according
to the protocols of the ethics committee of the Israeli Ministry of
Health (no.1221/19) and fulfill “The ARRIVE guidelines 2.0” for animal
research. The Collagen induced arthritis (CIA) model was performed as
previously described by us [32-34]: Bovine type II collagen
(Chondrex, Redmond, WA, USA) was emulsified 1:1 with mycobacterium
tuberculosis H37RA in Freund’s incomplete adjuvant (Difco Laboratories,
Detroit, MI, USA). DBA/1J males were subcutaneously injected into the
base of the tail with 100 μg emulsion. A boost injection of bovine type
II collagen in PBS, at the base of the tail, was given 21 days later.
Intraperitoneal injections of IVIG (OMRIX ltd, Rehovot, Israel), 3mg/0.1
ml/mouse, started at score of 2-3 after the boost injection and repeated
on a weekly base. PBS as vehicle (volume) and non-treated mice were used
as controls, n = 10 per each group. The mice were sacrificed after 48
days.
Arthritis scoring The clinical scores and the hind paw widths
of the mice were monitored daily over the course of each experiment. The
development of arthritis was assessed daily, and the severity of
arthritis was scored for each paw on a 3-point scale, in which
0 = normal appearance, 1 = localized edema/ erythema over one surface of
the paw, 2 = edema/ erythema involving more than one surface of the paw,
3 = marked edema/erythema involving the whole paw. The scores of all
four paws were added for a composite score, with a maximum score of 12
per mouse. Ankle thickness of the hind paws was measured in millimeters
(mm) at the widest point (the malleoli) with the legs fully extended
with digital calipers (Manostat, Herisau, Switzerland).
Histopathological
assessment
The paws of the mice were obtained from the sacrificed mice and fixed in
4% formalin (Sigma-Aldrich St Louis, MO, USA), decalcified, cut, and
stained with H&E. Samples were examined by light microscopy (×200
magnification). All histological evaluations performed by pathologists
were double blinded.