Blocking Kv1.3 potassium channels prevents postoperative neuroinflammation and cognitive decline without impairing wound healing in mice

Abstract

Background: Postoperative cognitive decline (PCD) requires microglial activation. Voltage-gated Kv1.3 potassium channels are involved in microglial activation. We determined the role of Kv1.3 in PCD and the efficacy and safety of inhibiting Kv1.3 with phenoxyalkoxypsoralen-1 (PAP-1) in preventing PCD in a mouse model.

Methods: After institutional approval, we assessed whether Kv1.3-deficient mice (Kv1.3^-/-) exhibited PCD, evidenced by tibial-fracture surgery-induced decline in aversive freezing behaviour, and whether PAP-1 could prevent PCD and postoperative neuroinflammation in PCD-vulnerable diet-induced obese (DIO) mice. We also evaluated whether PAP-1 altered either postoperative peripheral inflammation or tibial-fracture healing.

Results: Freezing behaviour was unaltered in postoperative Kv1.3^-/- mice. In DIO mice, PAP-1 prevented postoperative (i) attenuation of freezing behaviour (54 [17.3]% vs 33.4 [12.7]%; P=0.03), (ii) hippocampal microglial activation by size (130 [31] pixels vs 249 [49]; P<0.001) and fluorescence intensity (12 000 [2260] vs 20 800 [5080] absorbance units; P<0.001), and (iii) hippocampal upregulation of interleukin-6 (IL-6) (14.9 [5.7] vs 25.6 [10.4] pg mg^-1; P=0.011). Phenoxyalkoxypsoralen-1 neither affected surgery-induced upregulation of plasma IL-6 nor cartilage and bone components of the surgical fracture callus.

Conclusions: Microglial-mediated PCD requires Kv1.3 activity, determined by genetic and pharmacological targeting approaches. Phenoxyalkoxypsoralen-1 blockade of Kv1.3 prevented surgery-induced hippocampal microglial activation and neuroinflammation in mice known to be vulnerable to PCD. Regarding perioperative safety, these beneficial effects of PAP-1 treatment occurred without impacting fracture healing. Kv1.3 blockers, currently undergoing clinical trials for other conditions, may represent an effective and safe intervention to prevent PCD.

Keywords: Kv1.3; microglial activation; neuroinflammation; phenoxyalkoxypsoralen-1; postoperative cognitive decline; potassium channel; wound healing.

Fig 1

Timeline of studies. (a) Behavioural assessment: mice (wild-type, with diet-induced obesity [DIO] or Kv1.3 deficient [Kv1.3^–/–]) were trained in the trace fear-conditioning paradigm 30 min before induction of general anaesthesia with isoflurane. During general anaesthesia, surgery was performed on the tibia of the left hindlimb. Before surgical incision, wild-type and DIO mice received either i.p. phenoxyalkoxypsoralen-1 (PAP-1) 40 mg kg⁻¹ or vehicle (MIGLYOL) repeated every 12 h thereafter. After 3 days, freezing behaviour was tested in the same context as the training was performed. (b) Hippocampal neuroinflammation assessments: mice with DIO underwent tibial surgery during general anaesthesia. At the end of surgery, the mice received either PAP-1 or vehicle (MIGLYOL) i.p., and repeated 12 h later. At 24 h, the mice were killed and prepared for assessment of either hippocampal microglial activation by immunohistochemistry or hippocampal interleukin-6 (IL-6) by enzyme-linked immunosorbent assay. (c) Systemic inflammation assessment: wild-type mice underwent tibial fracture and internal fixation surgery during general anaesthesia. At the end of surgery, the mice received either PAP-1 or vehicle (MIGLYOL) i.p. At 6 h, the mice were killed and blood collected for analysis of plasma IL-6. (d) Wound-healing assessment: wild-type mice underwent tibial surgery during general anaesthesia. At the end of surgery, the mice received either PAP-1 or vehicle (MIGLYOL) i.p., and repeated every 12 h, thereafter until the third day. On the 10th day, the mice were killed and the left hindlimbs were removed for assessment of fracture callus.

Fig 2

Role of Kv1.3 in postoperative cognitive decline in the trace fear-conditioning (TFC) paradigm. (a–c) Cohorts of mice were trained in a TFC paradigm immediately before surgery and tested for freezing behaviour 3 days after surgery. (a) Wild-type mice (12–14 weeks old C57bl/6j) were randomised to three groups (n=8–9 per group) that received no surgery/vehicle, surgery/vehicle, or surgery/phenoxyalkoxypsoralen-1 (PAP-1). (b) Wild-type mice with diet-induced obesity (DIO) were randomised to three groups (n=8–10 per group) that received no surgery+vehicle, surgery+vehicle, or surgery+PAP-1. (c) Mice deficient in Kv1.3 (Kv1.3^–/–) were randomised to two groups (n=9–10 per group) that received either sham (no surgery) or surgery. After wound closure and every 12 h thereafter, the mice randomised to PAP-1 received 40 mg kg⁻¹ i.p. vehicle consisted of MIGLYOL in the same volume as PAP-1. On the third day, freezing behaviour was tested in the same context as the training. (a) and (b) Analysed by one-way analysis of variance followed by Bonferroni post hoc test. (c) Analysed by unpaired t-test. ∗P=0.029; ∗∗P=0.005; §P=0.011; §§P=0.030.

Fig 3

Phenoxyalkoxypsoralen-1 (PAP-1) attenuates surgery-induced microglial activation. Mice with diet-induced obesity (DIO) were randomised to three groups (n=8 per group) that received neither surgery nor PAP-1 (DIO+sham), surgery alone with no PAP-1 (DIO+surgery), or surgery together with PAP-1 (DIO+surgery+PAP-1). After surgery, mice not randomised to receive PAP-1 (DIO+sham; DIO+surgery) were administered MIGLYOL, and mice randomised to PAP-1 received doses intraoperatively and 12 h postoperatively. At 24 h after surgery, mice were killed, and brains were perfused, fixed, and sectioned. Coronal sections (35 μm thick) from the dentate gyrus were stained with anti-Iba1 antibody. Immunofluorescence was performed with Alexa Fluor 488-labelled anti-rabbit antibody. (a) Representative photomicrographs from each of the groups. The upper panel represents DAPI staining, the middle panel staining for Iba1, and the lower panel a higher magnification. The internal scale marker represents 50 μm. (b) Iba1+ cells were counted manually by visual inspection of hippocampal sections of anatomically matched photomicrographs, with an average taken from four sequential sections per mouse. (c) To analyse microglial cell-body size (in pixels), each section was processed in a systematic way to create a binary image by applying a previously established threshold. (d) Iba1 fluorescence intensity (absorbance unit [AU]) was similarly measured with ImageJ from images captured using identical exposure times that also avoided saturating pixel intensities. Data (mean [standard deviation]) were analysed by one-way analysis of variance followed by Bonferroni post hoc test. ∗P=0.0012; ∗∗P<0.001.

Fig 4

Phenoxyalkoxypsoralen-1 (PAP-1) prevents surgery-induced hippocampal inflammation. Mice with diet-induced obesity were randomised to four groups (n=8 per group) that received either no surgery or surgery, and either PAP-1 or vehicle. At 24 h after surgery, the animals were killed and hippocampi were removed and assayed for interleukin-6 (IL-6) by enzyme-linked immunosorbent assay. Data were analysed by one-way analysis of variance followed by Bonferroni post hoc test. ∗P<0.001; ∗∗P=0.011.

Fig 5

Phenoxyalkoxypsoralen-1 (PAP-1) does not affect surgery-induced peripheral inflammation. Wild-type mice were randomised to four groups (n=4 per group) that received either no surgery+vehicle, no surgery+PAP-1, surgery+vehicle, or surgery+PAP-1. At 6 h after surgery, the animals were killed and blood removed for measurement of plasma interleukin-6 (IL-6) by enzyme-linked immunosorbent assay. Data were analysed by one-way analysis of variance followed by Bonferroni post hoc test. ∗P<0.001.

Fig 6

Phenoxyalkoxypsoralen-1 (PAP-1) does not affect fracture healing. Wild-type mice were randomised to two groups (n=10 per group) that received either PAP-1 or vehicle (control) administered intraoperatively and at 12 h intervals for 3 days. At 10 days after surgery, the mice were killed and the left tibiae were removed for stereological evaluation of fracture healing. (a) Vehicle control and (b) PAP-1 treated are representative histological images of fracture callus stained with Hall–Brunt's quadruple stain that illustrates bone tissue (red) and cartilage tissue (blue). The scale bar is 1 mm. (c) Volume of bone and cartilage tissue in the total fracture callus volume. (d) Percentage composition of bone and cartilage tissue in the fracture callus. Data were analysed by two-way analysis of variance followed by Sidak's post hoc test.