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Physostigmine Restores Impaired Autophagy in the Rat Hippocampus after Surgery Stress and LPS Treatment

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Abstract

Tissue damage and pathogen invasion during surgical trauma have been identified as contributing factors leading to neuroinflammation in the hippocampus, which can be protected by stimulation of the cholinergic anti-inflammatory pathway using the acetylcholinesterase inhibitor physostigmine. Macroautophagy, an intracellular degradation pathway used to recycle and eliminate damaged proteins and organelles by lysosomal digestion, seems to be important for cell survival under stress conditions. This study aimed to examine the role of autophagy in physostigmine-mediated hippocampal cell protection in a rat model of surgery stress. In the presence or absence of physostigmine, adult Wistar rats underwent surgery in combination with lipopolysaccharide (LPS). Activated microglia, apoptosis-, autophagy-, and anti-inflammatory-related genes and -proteins in the hippocampus were determined by Real-Time PCR, Western blot and fluorescence microscopy after 1 h, 24 h and 3 d. Surgery combined with LPS-treatment led to microglia activation after 1 h and 24 h which was accompanied by apoptotic cell death after 24 h in the hippocampus. Furthermore, it led to a decreased expression of ATG-3 after 24 h and an increased expression of p62/ SQSTM1 after 1 h and 24 h. Administration of physostigmine significantly increased autophagy related markers and restored the autophagic flux after surgery stress, detected by increased degradation of p62/ SQSTM1 in the hippocampus after 1 h and 24 h. Furthermore, physostigmine reduced activated microglia and apoptosis relevant proteins and elevated the increased expression of TGF-beta1 and MFG-E8 after surgery stress. In conclusion, activation of autophagy may be essential in physostigmine-induced neuroprotection against surgery stress.

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Fig. 1: Physostigmine reduced microglia activation after surgery stress in the hippocampus.
Fig. 2: Physostigmine reversed the enhanced ratio of Bax/Bcl-2, cytochrome c release and cleaved Caspase-3 and PARP after surgery stress in the hippocampus.
Fig. 3: Physostigmine reduced apoptotic cell death after surgery stress in the hippocampus.
Fig. 4: Physostigmine in combination with surgery stress increased the gene and protein expression of ATG-3 and Beclin-1 in the hippocampus.
Fig. 5: Physostigmine enhanced the protein expression of LC3-II in the hippocampus.
Fig. 6: Physostigmine reversed the accumulation of p62/SQSTM1 after surgery stress in the hippocampus.
Fig. 7: Physostigmine in combination with surgery stress enhanced expression of TGF-beta1 and MFG-E8 after surgery stress in the hippocampus.

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Acknowledgments

We thank Marie-Christin Gaerz and Rosalie Schmidt for excellent technical assistance.

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Authors and Affiliations

  1. Department of Anesthesiology and Operative Intensive Care Medicine (CCM, CVK), Charité-Universitätsmedizin Berlin, corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Campus Virchow-Klinikum, Augustenburger Platz 1, 13353, Berlin, Germany

    Clarissa von Haefen, Marco Sifringer, Alexander Kalb, Adrián González-López, Annalena Tegethoff, Nadine Paeschke & Claudia D. Spies

  2. Department of Neonatology, Charité-Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany

    Stefanie Endesfelder

  3. CIBER-Enfermedades Respiratorias, Instituto de Salud Carlos III, Madrid, Spain

    Adrián González-López

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  1. Clarissa von Haefen
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  2. Marco Sifringer
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  3. Stefanie Endesfelder
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  4. Alexander Kalb
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  7. Nadine Paeschke
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  8. Claudia D. Spies
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Correspondence to Clarissa von Haefen.

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All authors have read the journal’s policy on disclosure of potential conflicts of interest and have none to declare.

Ethical Approval

All animal experiments were approved and performed in accordance with the guidelines of the Charité-Universitätsmedizin Berlin, Germany and the national ethic principles (registration no. G 0253/09).

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von Haefen, C., Sifringer, M., Endesfelder, S. et al. Physostigmine Restores Impaired Autophagy in the Rat Hippocampus after Surgery Stress and LPS Treatment. J Neuroimmune Pharmacol 13, 383–395 (2018). https://doi.org/10.1007/s11481-018-9790-9

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