The present study demonstrated that sepsis induced by CLP resulted in behavioral deficits in open field, elevated plus maze and forced swimming tasks 10 days after surgery that mainly resolved within 30 days. The main finding of the study is that treatment with immunoglobulin after sepsis induction caused earlier resolution in locomotor function and behavioral alterations including depression and anxiety. All surviving animals presented nearly normal performance in all tests performed after 60 days indicating recovery of functional disturbances. The overall survival rate in the CLP group was 50% in accordance with other studies, whereas it was 70% after the standard IgG and 80% in the Ig GAM treatment groups.
Long-term cognitive impairment including alterations in memory, anxiety, depression and impairment in executive function was reported in sepsis survivors . Each of these was shown to be associated with a decreased quality of life. CLP model was extensively used to address the mechanisms associated with cognitive deficits seen in sepsis and help to investigate therapeutic approaches for this problem [3, 15]. Among cognitive functions, memory was reported to be the most frequently observed deficit. The present study evaluated the changes in locomotor activity, anxiety and depressive-like symptoms in sepsis-surviving rats.
We studied three basic behavioral tasks that were mainly used in those experiments determining long-term effects in sepsis-surviving animals. Forced swimming test was used as a model for determining depressive-like behavior , elevated plus maze test was used for anxiety measurement , and animals were put into different open field facilities to determine the alterations in locomotor and functional activities . Time-dependent measurements in CLP-induced septic animals were in accordance with previous reports in terms of decreases in locomotor activity and increases in depressive-like behavior . Rearing and grooming events dramatically decreased in animals in the CLP group, which indicated attenuation in the locomotor activity by the induction of sepsis. The activity was attenuated on day 10 and 30. These results were in contrast with the previous reports, which indicated no change in the locomotor and exploratory activities in the open field test in the CLP group compared with control group, 10 days after the procedure [3, 8, 15–17]. Similar to these, Leite et al. found no significant effects of CLP on the locomotor activity in OFT 7 days after surgery compared with the sham group . Overall, both immunoglobulin treatments were able to elevate the number of rearing and grooming events compared with the CLP group on day 10, whereas the IgGAM group was also able to increase grooming on day 30. Although there was no difference in terms of the time spent in the central zone when the CLP group compared with the control group, the IgGAM group stays longer than CLP group in the central zone on day 10, which indicated a better reversal of anxiety-like behavior.
In the FST, the time of immobility, indicating a depressive-like behavior, was higher in the CLP group compared with the control group on day 10, which is compatible with previous reports that used the CLP model. Two previous studies reported a significant increase in the immobility time in the CLP group compared with the sham group, 10 days after CLP procedure [5, 15]. In addition, a recent study that used a sucrose consumption test as a depressive-like state model reported that CLP induction caused anhedonia with the reduced sucrose intake compared with the sham group, 17 days after surgery . We found no differences in the time of immobility between the groups on day 30 and 60 meaning that depressive-like state was resolved before 30 days in the CLP group. In a previous study, the CLP group was shown to have increased immobility time compared with the sham group on day 10 and day 30, but not on day 60 . We showed that both IgG and IgGAM treatments decreased immobility time compared with the CLP group on day 10, which can be interpreted that both treatments reversed CLP-induced depressive-like behaviors by day 10.
The CLP procedure produced an anxiogenic-like effect 10 days after surgery. This was demonstrated by the decreased time spent in open and closed arms of the EPM task. This was not the case in the previous experimental trials that studied long-term depression and anxiety after sepsis. Two experimental trials demonstrated no change in the anxiety-like behavior after CLP [3, 5]. Most recently, Leite et al. demonstrated significant anxiety in CLP-induced septic animals, which is in accordance with our findings . In the present study, anxiety-like behavior had resolved in the CLP group by day 30 after surgery. Both immunoglobulin treatments increased the time spent in open arms, which was coupled with higher number of entries to open arms compared with the CLP group on day 10. Treatment with IgG and IgGAM caused early resolution of functional and behavioral alterations after sepsis induction, which indicated some treatment effect. This was parallel to the improved survival in the treatment groups. The underlying mechanisms of which immunoglobulin exerts these effects are most probably due to their regulatory involvement in the inflammatory processes causing endothelial activation and the breakdown of the BBB . In our previous experimental trial, immunoglobulin treatment protected the brain from sepsis-induced effects . Fewer morphologic changes and less disruption in BBB integrity were evident, which was in accordance with the improved survival in the treatment groups. The BBB failure in sepsis is explained by endothelial cell injury through cytokines such as TNF-α and IL-β that leads to upregulation of endothelial surface antigens and subsequent white blood cells adherence as well as microcirculatory dysfunction . Experimental studies have shown that IVIG is capable of significantly reducing leukocyte adhesion and by normalizing capillary perfusion attenuate microcirculatory dysfunction . Using intravital microscopy, the effects of immunoglobulins on leukocyte recruitment in superficial brain vessels were visualized. Immunoglobulins potentially reduced leukocyte rolling and adhesion in experimental autoimmune encephalomyelitis (EAE) . It has been suggested that immunoglobulins exert these actions either through their effects on cytokine production or directly by reducing cell adhesion molecule production [22, 23]. Experiments using cultures of bovine and human brain endothelium suggest that cytokines increase BBB permeability. TNF-α was stained on microvascular vessels of the brain in sepsis and recombinant TNF-α also increased the permeability in mice . Studies demonstrated that immunoglobulins contain high-affinity neutralizing antibodies against IL-1 IL-6 and TNF-α and downregulate their synthesis by their effects on T cells [21, 24].
The beneficial effects of IV immunoglobulins on the BBB are most probably due to their regulating effects on the complement system . The neurotoxic effects of complement fragments have recently been studied and blocking C5a anaphylatoxin resulted in neuroprotective effects in CLP-induced septic animals . Arumugam et al. have demonstrated that complement activation in neuroinflammation was regulated with immunoglobulins in an experimental stroke model, and immunoglobulins reduced the mortality and the amount of brain damage .
In the current study, two different types of immunoglobulin preparations were used expecting that IgM would improve functional and behavioral alteration caused by sepsis. Early experimental studies have demonstrated superior effects of IgM in comparison with IgG in terms of their activities on inflammation [28, 29]. Both intravenous IgM and IgG preparations markedly attenuated the endotoxin-induced leukocyte adherence; however, only intravenous IgM was capable of further reducing venular leukocyte adherence, whereas IgG did not show protective effects compared with controls. This effect was also evident with the measurement of functional capillary density (FCD) where IgM significantly ameliorated the LPS-induced decrease of FCD after 24 h of endotoxemia . Protective effects of IgM on tissue integrity were studied on lungs where significantly reduced alveolar damage was evident parallel with the histological evaluation . This study showed a better survival rate with IgM-enriched immunoglobulin administration during sepsis; however, both immunoglobulin preparations were able to reverse behavioral deficits within 10 days, which were resolved by 60 days after surgery in all groups.
One limitation of the study might be considered as the possible cross-species binding differences since we used human IV immunoglobulin preparation in rats as rodents that are routinely used as a convenient first-line model for clinical evaluation of IVIG therapies. Studies on the mapping of the binding side on human immunoglobulins suggested that the Fc interactions are, in some respects, very similar across species . Second, the timing of IVIG administration might be considered as too early as to extrapolate its impact for clinical effectiveness; however, similar timing of IVIG administration has been widely used in experimental studies testing the potential of therapeutic effects. Third, in our setup, we were not able to evaluate a memory deficit which was reported to be the most frequently observed deficit in humans.