2.1. Stereotactic Intrahippocampal Injection in Vivo

Before mounting on a stereotactic frame (Narishige, Tokyo, Japan), 2-month-old female Wistar rats (Charles River Laboratories GmbH, Sulzfeld, Germany) were anesthetized with S-ketamine (100 mg/kg IP (intraperitoneal)) and xylazine (15 mg/kg IP). The injection sites were calculated relatively to the bregma (+ 5.2 mm posterior, ± 4.3 mm lateral, + 4.8 mm deep), as previously reported [19,20,21]. After the trepanation, bilateral injection of 5 µL human non-diluted CSF (into each hippocampus) was performed using a Hamilton syringe (75 N, Hamilton AG, Bonaduz, Switzerland) in 10 steps of 0.5 µL every two min. After the last aliquot of 0.5 µL, the syringe was left in situ for another two minutes. CSF samples containing anti-GAD were obtained from two patients (both female, 18 years old and 27 years old) with temporal lobe epilepsy due to limbic encephalitis. The GAD-antibodies were detected by indirect immune-techniques by congruent GAD65 specific signals on mouse brains, transfected cells and immune-dot-blot. Titration was done on cell-based assays in dilution steps of 1:2 and multiples (see [19]). The procedure was described by Christian G. Bien [21,22]. The titers were 1:1000 (anti-GAD A) and 1:250 (anti-GAD B). As control groups, we injected either a CSF sample from a patient with epilepsy (absence of anti-GAD) or a saline solution (NaCl 0.9%). In order to control for the surgery, field potential recordings were also performed in non-operated, naive animals as a third control group (Table 1). Since we only had a small sample of anti-GAD-A, experiments II and III were carried out only with anti-GAD-B. Following postoperative pain management (metamizole 100–150 mg/kg), the rats were allowed to recover in an atmosphere with enhanced oxygen fraction (4–5 L/min in an 8 L glass vessel).

Experimental groups. Number of slices is given in parentheses.

REDs: recurrent epileptic discharges, ACSF: artificial cerebrospinal fluid.

To confirm the diffusion of the injected agents (CSF samples containing anti-GAD A and B, anti-GAD-negative human CSF and saline solution) as previously reported [20], we performed experiments using an injection of an immunofluorescent marker dye. After injection, cryostat sections of the brain were covered with ProLong® Gold antifade reagent with DAPI (Invitrogen/ThermoFisher Scientific, Carlsbad, CA, USA). Evaluation was done using the Leica DMI6000B microscope and LAS AF software (Figure 1).

Marker dispersion in the hippocampus. The immunofluorescence image shows that the injected marker disperses from the injection site (CA3-region) into the CA1-region. (injection site denoted by the red +). CA = Cornu Ammonis.