5.3.1. Effect of surgery and anaesthesia on bodyweight
Surgery, sham surgery and anaesthesia produced a decrease in body weight. Two-way repeated measure ANOVA of bodyweight showed an effect of time [F (3,99) =101, P< 0.001], an effect of procedure [F (3,33) = 4.432; P=0.01] and a procedure x time interaction [F (9,99) =11.34, P<0.001]. Post hoc comparisons revealed that anaesthesia decreased bodyweight at 24 h [P<0.05], 48 h [P<0.001] and 72 h [P<0.001] when compared to home cage controls. Sham surgery decreased bodyweight when compared to home cage controls at 24 h [P<0.05], 48 h [P<0.001] and 72 h [P<0.01]. PBS administration decreased bodyweight when compared to home cage controls at 24 h [P<0.01], 48 h [P<0.001], 72 h post-surgery [P<0.001]. All groups lost weight but there were no weight differences between the sham/complete surgery and the anaesthesia treated groups (Fig 4.4.A).
5.3.2. Effect of surgery and anaesthesiain behavioural tasks
Two-way repeated measure ANOVA of saccharin preference showed an effect of time [F (2,62) = 6.861, P= 0.0020], no effect of procedure [F (3,31) = 2.849, P= 0.0535], but a procedure x time interaction [F (6,62) = 3.924, P= 0.0022]. Post hoc comparisons revealed that anaesthesia, sham surgery and PBS administration decreased the saccharin preference over the 24 h period [P<0.05], and from 24 h-48 h after surgery [P<0.01] compared to home cage controls (Fig 4.4.B). Two-way repeated measure ANOVA of total fluid consumption showed an effect of time [F (2,62) = 11.87, P<0.001], no effect of procedure [F (3,31) = 0.3587, P= 0.7832], no procedure x time interaction [F (6,62) = 0.916, P= 0.4857] (data not shown). Two-way repeated measure ANOVA of saccharin consumption showed an effect of time [F (2,62) = 3.219, P = 0.0468], no effect of procedure [F (3,31) = 1.380, P= 0.2672], no procedure x time interaction [F (6,62) = 1.078, P= 0.3852] (data not shown). Two-way repeated measure ANOVA of water consumption showed an effect of time [F (2,62) = 12.98, P<0.001], an effect of procedure [F (3,31) = 3.303, P= 0.0331], a procedure x time interaction [F (6,62) = 2.284, P= 0.0468]. Post hoc comparisons revealed that sham surgery increased water consumption over the 24 h period [P<0.05] and that sham surgery [P<0.001], complete surgery and anaesthesia [P<0.05] increased water consumption from 24 to 48 h after injection compared to home cage controls (Fig 4.4.C).
Two-way repeated measure ANOVA of time spent grooming (back licking) showed an effect of time [F (1,28) = 51.05, P<0.001], an effect of procedure [F (3,28) = 5.496, P<0.001], and a
157 procedure x time interaction [F (3,28) = 3.762, P= 0.0218]. Post hoc comparisons revealed that anaesthesia [P<0.01], sham surgery and PBS administration [P<0.001] increased grooming duration 5-10min following sucrose application 48 h after surgery compared to the home cage controls (Fig 4.4.D).
One way ANOVA of percentage of time spent in the peripheral area in the open field showed no effect of procedure [F (3,32) =2.599, P=0.0693] or total distance travelled in the OF [F (3,34) =1.142, P=0.3461] compared to home cage controls (data not shown).
One-way ANOVA of immobility in the TST showed no effect of procedure [F (3,35) = 2.374, P= 0.0868] (Fig 4.4.E). Two-way repeated measure ANOVA of immobility in the FST showed no effect of time in the FST [F (1,32) =1.337, P= 0.2561], no effect of procedure [F (3,32) =1.895, P= 0.1504], and no procedure x time interaction [F (3,32) = 0.1446; P=0.9324] compared to home cage controls (Fig 4.4.F), indicating that neither surgery nor anaesthesia affected immobility in the TST or FST.
5.3.3. Effect of surgery and anaesthesia on GFAP and Iba1 immunoreactivity in the
PLC
Post mortem analysis of GFAP immunoreactivity in the PLC is shown in Fig 4.5.A.One way ANOVA of GFAP immunoreactivity showed an effect of procedure [F (3,35) =30.74, [P<0.001]. Post hoc comparisons revealed that PBS injection increased GFAP immunoreactivity in the PLC when compared to home cage control [P<0.001] 72 h post- surgery. These results suggest that surgery (with micro capillary injection of PBS) but not anaesthesia increased GFAP immunoreactivity in the PLC.
Analysis of Iba1 immunoreactivity in the PLC is shown in Fig 4.5.B. One-way ANOVA of Iba1 immunoreactivity showed an effect of procedure [F (3,8) =6.254, P=0.0171]. Post hoc comparisons revealed that PBS administration increased Iba1 immunoreactivity when compared to home cage controls [P<0.01] 72 h post-surgery. These results suggest that surgical placement of the microcapillary and/or delivery of PBS, but not anaesthesia or surgical manipulation apart from the injection procedure, increased Iba1 immunoreactivity.
158
Figure 4.4: Investigation of the impact of surgery and anaesthesia on behaviour
Controls were used to assess the effect of the anaesthesia and surgery on bodyweight compared to PBS and L- AAA-treated groups. Surgery (PBS), sham surgery or anaesthesia decreased the bodyweight. (Home cage; N=10, Avertin; N=10, Sham surgery; N=9; PBS; N=8), A), reduced saccharin preference B) and increased water consumption C) (Home cage; N=9, Avertin; N=10, Sham surgery; N=8; PBS; N=8).Surgery and anaesthesia increased grooming behaviour indicating by back licking duration (Home cage; N=8, Avertin; N=9, Sham surgery; N=8; PBS; N=7) D). There was no differences between the groups on immobility in the TST (Home cage; N=10, Avertin; N=10, Sham surgery; N=10; PBS; N=9) E) or the FST (Home cage; N=10, Avertin; N=10, Sham surgery; N=9; PBS; N=7) F). Data are expressed as mean ± SEM, *, P<0.05; **, P<0.01, ***, P<0.001 for all groups versus home cage controls.
159
Figure 4.5: Investigation of the impact of surgery andanaesthesia on GFAP and Iba1 immunoreactivity in the PLC
Controls were used to assess the impact of the anaesthesia and surgery on both behaviour and GFAP and Iba1 immunoreactivity in the PLC. A) Surgery increased GFAP immunoreactivity in the PLC 72 h post L-AAA administration (Home cage; N=10, Avertin; N=10, Sham surgery; N=10; PBS; N=9). B) Surgery increased Iba1 immunoreactivity 72 h post L-AAA administration (Home cage; N=3, Avertin; N=3, Sham surgery; N=3; PBS; N=3). Data are expressed as mean ± SEM, *, P<0.05; **, P<0.01, ***, P<0.001 versus home cage controls.
160
6.
Discussion
The overarching aim of this study was to refine a mouse model of cortical astrocytic impairment leading to depressive-related behaviours. Results indicate that while a depressive behavioural phenotype and reduced cortical GFAP immunoreactivity are observed following two consecutive daily doses of L-AAA (50μg/ul; 1μl; 2 days), a single dose (50μg/ul; 1μl) was also sufficient to induce both depressive-like behaviours and reduced GFAP immunoreactivity. Following L-AAA administration, a transient depressive-like behaviour and reduced cortical GFAP immunoreactivity was evident over a 72 h post administrative period leading to a recovery from behavioural deficit and reduced GFAP immunoreactivity over 7 days. In addition to reduced GFAP, a reduction in the immunoreactivity of the astrocytic protein s100b was observed in the PLC following a single dose of L-AAA to the PLC without affecting NeuN or Iba1 immunoreactivity.