Graphical abstract

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   [41]Open in a new tab

Highlights

     * •
       The neurotoxicity of lidocaine is associated with age and the
       frequency of exposure
     * •
       Repeated lidocaine exposure leads to synaptic and cognitive
       impairment in aged mice
     * •
       Repeated lidocaine exposure activates microglia and neurotoxic A1
       astrocytes
     * •
       The NF-κB pathway plays a crucial role in neurotoxicity induced by
       lidocaine
     __________________________________________________________________

   Pharmacology; Neuroscience; Immunology; Cell biology

Introduction

   With the aging of the global population, it is estimated that up to 50%
   of elderly individuals will undergo one or more surgical procedures
   worldwide.[42]^1 Research has demonstrated that elderly patients with a
   range of conditions, such as intrahepatic and extrahepatic biliary
   stones, urolithiasis, bladder tumors, benign prostatic hyperplasia, and
   emergency trauma repair are susceptible to undergoing multiple
   surgeries within a limited time frame.[43]^2^,[44]^3^,[45]^4^,[46]^5
   Postoperative cognitive dysfunction (POCD) is a common neurological
   complication that often manifests after surgery and anesthesia,
   especially in the elderly population.[47]^6 POCD is characterized by
   either temporary or permanent cognitive decline, memory impairments,
   and difficulty adjusting to social situations, which potentially
   results in permanent disability and increased mortality of
   patients.[48]^7^,[49]^8 The pathogenesis of POCD remains uncertain but
   is highly likely to be linked with the age of patients, the type of
   surgical operations, and the administration of
   anesthesia.[50]^9^,[51]^10 Hence, when treating elderly patients,
   particular attention should be given to preventing anesthesia-induced
   cognitive dysfunction, considering the frequency of anesthesia and the
   amount and duration of perioperative anesthetics administered.

   Lidocaine is a commonly used local anesthetic with various
   applications, such as surface anesthesia, infiltration anesthesia,
   nerve blocks, and intrathecal anesthesia.[52]^11 Recently, intravenous
   lidocaine has been widely used in the perioperative setting to
   alleviate postoperative pain and minimize opioid usage,[53]^12 speed up
   gastrointestinal function recovery,[54]^13 and even potentially improve
   tumor prognosis.[55]^14 However, it is important to recognize that
   systemic lidocaine has the potential to induce central nervous system
   (CNS) toxicity, which may lead to a variety of neurological
   complications, including oversedation, drowsiness, cognitive
   alterations, and, in severe cases, seizures, coma, and potentially
   death.[56]^15^,[57]^16 Previous studies from various
   laboratories,[58]^17^,[59]^18^,[60]^19 including our own
   research,[61]^20 have shown that lidocaine can induce damage to neurons
   in the brain, spinal cord, and peripheral nerves in a time- and
   dose-dependent manner. Specifically, several animal studies suggest
   that the administration of lidocaine may induce neuroinflammation,
   which potentially involves the activation of immune
   cells.[62]^21^,[63]^22 Moreover, the neurotoxic effects are
   significantly amplified with repeated exposure to anesthetics compared
   to a single exposure.[64]^23^,[65]^24^,[66]^25 Given the frequent
   necessity of multiple anesthesia and surgeries for elderly individuals
   to prevent recurring illnesses and uphold health, it is vital to
   investigate the effects of repeated lidocaine exposure on
   neurocognitive function at therapeutic levels, along with the
   underlying mechanisms.

   Astrocytes, the most abundant cell type in the brain, play crucial
   roles in maintaining brain homeostasis and are involved in the
   neuroinflammatory response.[67]^26 In response to CNS injury and
   disease, astrocytes undergo changes in morphology and function,
   transforming into reactive cells that exhibit either a neurotoxic A1
   phenotype or a neuroprotective A2 phenotype.[68]^27 Recent studies have
   shown that microglia in a classically activated state are capable of
   inducing the formation of reactive A1 astrocytes through the secretion
   of interleukin-1 alpha (IL-1α), tumor necrosis factor-alpha (TNF-α),
   and complement component 1q (C1q).[69]^28 Neurotoxic A1 astrocytes
   induced by microglial activation have been proposed as a significant
   contributor to age-related neurodegenerative disorders[70]^29 and the
   early onset of POCD.[71]^30 Therefore, further validation is required
   to investigate the effects of lidocaine exposure on microglial and
   astrocyte responses that are considered predominant neuropathological
   hallmarks of POCD.

   Characterizing the dose- and age-related effects of intravenous
   lidocaine exposure is critical for creating safe clinical practices and
   providing informed preoperative counseling. Thus, the present study was
   performed to investigate the effects of different frequencies of
   lidocaine exposure (single vs. repeated) on neurocognitive function in
   different age groups (4 vs. 18 months old) of mice. Specifically, we
   investigated changes in cognitive function, synaptic plasticity, and
   the responses of microglia and astrocytes following lidocaine exposure.
   Additionally, we employed transcriptome sequencing and bioinformatics
   analysis to explore the molecular mechanisms underlying the
   neuropathological changes observed in the hippocampus. Our findings
   reveal that compared to a single exposure, repeated exposure of aged
   mice to lidocaine is highly neurotoxic. The differentially expressed
   genes enriched in biological processes were mainly involved in
   inflammatory responses and synaptic function. Notably, our results
   suggest the potential therapeutic benefits of
   dehydroxymethylepoxyquinomicin (DHMEQ), a specific inhibitor of NF-κB,
   in mitigating the adverse effects of repeated lidocaine exposure on
   cognitive function in aged mice. Furthermore, the depletion of
   microglia with PLX5622 successfully prevented the activation of
   neurotoxic A1 astrocytes and synaptic loss, emphasizing the promising
   role of targeted interventions in safeguarding neuronal integrity in
   response to lidocaine-induced neurotoxicity.

Results

Convulsive dose, plasma concentration and hemodynamic changes after the
administration of lidocaine

   In this study, we aimed to determine the median convulsive dose (CD50)
   of intravenous lidocaine in aged mice, which was found to be 22.07 mg
   kg^−1. We also measured the plasma concentration of lidocaine to be
   7.42 ± 0.47 μg mL^−1 during the convulsive episodes. In the
   experimental group, the plasma concentration of lidocaine was assessed
   at 0 h, 1 h, 2 h, and 4 h after administering lidocaine using a bolus
   dose of 18.45 mg kg^−1 followed by a continuous infusion of 12.30 mg
   kg^−1 h^−1 for a duration of 2 h. The measured plasma concentrations at
   these time points were 4.35 ± 0.58 μg mL^−1, 1.24 ± 0.20 μg mL^−1,
   0.82 ± 0.08 μg mL^−1, and 0.09 ± 0.03 μg mL^−1, respectively
   ([72]Figure 1A). The results clearly indicated that the experimental
   group had a noticeably lower plasma concentration of lidocaine, which
   is clinically relevant, compared to the concentration observed during
   convulsion at every time point. Furthermore, no convulsive behavior was
   observed, and the heart rate (HR) and mean arterial blood pressure
   (MAP) remained stable throughout continuous intravenous infusion in the
   experimental group ([73]Figures 1A and 1B). These findings suggest that
   the administered dosage fell within the safe range. Accordingly, the
   dosage and administration route of lidocaine were selected as optimal
   for the subsequent studies.

Figure 1.

   [74]Figure 1
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   Plasma concentration and hemodynamic changes after the administration
   of lidocaine

   (A) Plasma concentrations of lidocaine following the administration of
   lidocaine (n = 5).

   (B) HR measured at baseline, immediately after administration, at
   1 min, 1 h, and 2 h post-administration of lidocaine (n = 5).

   (C) MAP measured at baseline, immediately after administration, at
   1 min, 1 h, and 2 h post-administration of lidocaine (n = 5).

Repeated lidocaine exposure triggers cognitive impairment in aged mice but
not adult mice

   We then confirmed the influence of different frequencies of intravenous
   lidocaine on cognitive function in mice of different ages. To do that,
   we established an animal model in which aged and adult mice were
   exposed to clinically relevant concentrations of lidocaine through
   single or repeated administration. The Morris water maze (MWM) test was
   utilized to evaluate cognitive function. Specifically, [76]Figure 2A
   displays the typical swimming paths of the aged mice on the fifth day
   of the place navigation and sixth day of the probe trial. During the
   training session, all three groups of aged mice showed decreased escape
   latency over time, indicating an active learning process. However, aged
   mice that were repeatedly exposed to lidocaine spent more time finding
   the hidden platform during training days 2–5 compared to the control
   and single lidocaine groups ([77]Figure 2B). During the probe trials,
   aged mice that were repeatedly exposed to lidocaine spent significantly
   less time in the target quadrant ([78]Figure 2C) and had fewer platform
   crossings ([79]Figure 2D) than the other two groups, indicating
   impaired memory performance. In addition, swim velocity was not
   significantly different among the groups ([80]Figure 2E), suggesting
   that the differences in the MWM test results were induced by variations
   in cognitive function.

Figure 2.

   [81]Figure 2
   [82]Open in a new tab

   Repeated lidocaine exposure causes cognitive impairments in aged mice
   but not adult mice

   (A) Representative swim paths of aged mice on the fifth day of the
   place navigation and sixth day of the probe trial in the MWM test.

   (B) Escape latency of aged mice to find the hidden platform during the
   5-day training trials.

   (C) Time spent in the target quadrant during the probe test.

   (D) Number of platform crossings of aged mice during the probe test.

   (E) Swim velocity of aged mice during the 5-day training trials.

   (F) Representative swim paths of adult mice on the fifth day of the
   place navigation and sixth day of the probe trial.

   (G) Escape latency of aged mice to find the hidden platform during the
   5-day training trials.

   (H) Time spent in the target quadrant during the probe test.

   (I) Number of platform crossings of adult mice during the probe test.

   (J) Swim velocity of adult mice during the 5-days training trials. Con:
   control group; Sin lido: single lidocaine exposure group; Rep lido:
   repeated lidocaine exposure group. n = 10 per group. ∗p < 0.05 compared
   with the con group, ^#p < 0.05 compared with the sin lido group. See
   also [83]Figure S1.

   To further examine the role of age in lidocaine-induced cognitive
   impairment, adult mice received single or repeated doses of lidocaine.
   The MWM test revealed that in all three groups, the escape latency
   ([84]Figure 2G), time spent in the target quadrant ([85]Figure 2H),
   number of target crossings ([86]Figure 2I), and swim velocity
   ([87]Figure 2J) were not significantly different. These data
   demonstrate that cognitive impairment in aged mice can result from
   repeated exposure to lidocaine, rather than a single exposure.
   Sevoflurane, an inhalation anesthetic, is widely used in clinical
   anesthesia and has been linked to cognitive impairment, particularly in
   the elderly.[88]^31 Consistent with prior research, mice in the Rep
   sevo group exhibited prolonged search time for the hidden platform and
   reduced time spent in the target quadrant compared to Con group. Our
   MWM test results further indicated that repeated exposure to lidocaine
   somewhat worsened the working memory impairment induced by sevoflurane
   in aged mice, as evidenced by the Rep Sevo+Lido group spending less
   time in the target quadrant and making fewer platform crossings during
   the probe trials compared to the Rep sevo group ([89]Figure S1).

RNA-seq analysis revealed that DEGs in the hippocampus were enriched in
inflammatory responses and synaptic signaling after repeated lidocaine
exposure in aged mice

   Next, we performed RNA-seq analysis to identify genes and molecular
   pathways underlying lidocaine-induced cognitive impairment. In the
   hippocampus of aged mice that were administered lidocaine repeatedly,
   we identified a total of 480 genes with differential expression (DEGs).
   Specifically, 281 DEGs (59%) were found to be significantly
   upregulated, while 199 DEGs (41%) were significantly downregulated
   ([90]Figures 3A and 3B). In contrast, adult mice repeatedly exposed to
   lidocaine did not show a significant difference in overall gene
   expression compared to the control group ([91]Figures 3C and 3D).

Figure 3.

   [92]Figure 3
   [93]Open in a new tab

   RNA-seq analysis indicated that DEGs in the hippocampus were enriched
   in inflammatory responses and synaptic signaling after repeated
   exposure to lidocaine

   (A) Volcano map of DEGs in aged mice. Red dots are upregulated genes,
   and blue dots are downregulated genes.

   (B) Heatmaps show the expression patterns of DEGs in aged mice.

   (C) Volcano map of DEGs in adult mice.

   (D) Heatmaps show the expression patterns of DEGs in adult mice.

   (E and F) Representative pathways enriched in the identified genes as
   determined by GSEA in the hippocampus of aged mice. The x axis
   represents the distribution of genes, from the most strongly
   upregulated to the most strongly downregulated.

   (G and H) KEGG pathway enrichment dot plot of the significantly
   upregulated and downregulated genes in the hippocampus of aged mice.
   Con: control group; Rep lido: repeated lidocaine exposure group. n = 3
   per group.

   After conducting the GSEA-GO analysis of the top and bottom NES
   rankings (top 20), it was noted that aged mice exposed to lidocaine
   repeatedly showed a notable increase in the acute inflammatory response
   ([94]Figure 3E) and a considerable decrease in synaptic structure and
   activity ([95]Figure 3F) in the hippocampus when compared to the
   control group. Furthermore, the significantly upregulated DEGs in the
   KEGG pathway analysis were found to be enriched in functional
   annotation related to tumor necrosis factor (TNF) and nuclear factor
   kappa B (NF-κB) signaling pathways ([96]Figure 3G). Additionally, KEGG
   pathway analysis revealed that a majority of the downregulated DEGs
   were enriched in axon guidance and cholinergic synapses in the aged
   mice hippocampus 24 h after repeated exposure to lidocaine
   ([97]Figure 3H). These data suggested that aged mice repeatedly exposed
   to lidocaine exhibited cognitive impairment, which was highly
   attributed to neuroinflammation and synaptic dysfunction in the
   hippocampus.

Repeated lidocaine exposure induces microglial activation and upregulates the
NF-κB pathway in the hippocampus of aged mice

   We utilized Iba-1 immunofluorescence to track microglial activation. In
   [98]Figure 4A, microglial cells in the hippocampus of control and
   single lidocaine-treated mice displayed smaller cell bodies with fewer
   and scattered processes. Conversely, microglia exhibited a reactive
   morphology in the hippocampus of mice repeatedly treated with
   lidocaine, characterized by enlarged cell bodies and thicker processes.
   Moreover, there was a noticeable increase in the number of Iba-1^+
   microglia in the hippocampus of aged mice following repeated lidocaine
   exposure compared to the control and single lidocaine groups. Western
   blot analysis further revealed an elevation in the levels of p-IκBα and
   p-NF-κB p65 in the hippocampus of aged mice after repeated lidocaine
   exposure, suggestive of NF-κB signaling activation ([99]Figures 4C–4E).
   Additionally, the expression levels of the microglia-derived
   proinflammatory cytokines IL-1α, TNF-α, and C1qA were significantly
   higher in the repeated lidocaine group compared to both the control and
   single lidocaine groups ([100]Figures 4F–4I).

Figure 4.

   [101]Figure 4
   [102]Open in a new tab

   Repeated lidocaine exposure induces microglial activation, upregulates
   the NF-κB pathway, and increases proinflammatory cytokines in the
   hippocampus of aged mice

   (A and B) Representative pictures of immunofluorescence staining of
   microglia (labeled by Iba-1 in green) and cell nuclei (labeled by DAPI
   in blue) in the hippocampal CA1 region. The statistical chart presents
   the number of microglia per field for each group (n = 6, scale bar:
   100 μm).

   (C–E) Representative western blot of p-IκBα, IκBα, p-NF-κB p65, and
   NF-κB p65 proteins in the hippocampus. Densitometric analyses of the
   immunoblots were performed, and the results are expressed as
   percentages relative to the control group (n = 4).

   (F–I) Representative western blot of proinflammatory cytokine IL-1α,
   TNF-α and C1qA proteins in the hippocampus. Densitometric analyses of
   the immunoblots were performed, and the results are expressed as
   percentages relative to the control group (n = 4). Con: control group;
   Sin lido: single lidocaine exposure; Rep lido: repeated lidocaine
   exposure. ∗p < 0.05 compared with the con group, ^#p < 0.05 compared
   with the sin lido group. Data were presented as the mean ± SD.

Repeated lidocaine exposure promotes the conversion of astrocytes to the A1
phenotype in the hippocampus of aged mice

   We examined the presence of A1 astrocytes, identified by glial
   fibrillary acidic protein (GFAP) and complement component 3 (C3), as
   well as A2 astrocytes, marked by GFAP and S100A10, in the hippocampus
   of aged mice following lidocaine exposure. Immunofluorescence analysis
   revealed a significant increase in the number of C3/GFAP-positive
   astrocytes in the hippocampus after repeated lidocaine exposure
   compared to the control and single lidocaine groups ([103]Figures 5A
   and 5B). Conversely, the number of S100A10/GFAP-positive astrocytes in
   the repeated lidocaine-treated group was significantly lower than that
   in the control and single lidocaine-treated groups ([104]Figures 5C and
   5D). Furthermore, RT‒qPCR results showed a significant increase in the
   levels of A1-specific genes (C3, H2-D1, Serping1, Amigo2) and a
   decrease in the levels of A2-specific genes (S100A10, CD14, Emp1,
   Stat3) in the hippocampus of aged mice after repeated lidocaine
   exposure ([105]Figure S2).

Figure 5.

   [106]Figure 5
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   Repeated lidocaine exposure promotes the transformation of astrocytes
   from the A2 phenotype to the A1 phenotype in the hippocampus of aged
   mice

   (A and B) Representative pictures of immunofluorescence staining of
   astrocytes (labeled by GFAP in green), neurotoxic markers (labeled by
   C3 in red) and cell nuclei (labeled by DAPI in blue) in the hippocampal
   CA1 region. The statistical chart presents the number of
   C3/GFAP-positive cells per field for each group (n = 4, scale bar:
   50 μm).

   (C and D) Representative pictures of immunofluorescence staining of
   astrocytes (labeled by GFAP in green), a neuroprotective marker
   (labeled by S100A10 in red) and the cell nucleus (labeled by DAPI in
   blue) in the hippocampal CA1 region. The statistical chart presents the
   number of S100A100/GFAP-positive cells per field for each group (n = 4,
   scale bar: 50 μm).

   (E and F) Representative pictures of immunofluorescence staining of
   astrocytes (labeled by GFAP in green), BDNF (red) and cell nuclei
   (labeled by DAPI in blue) in the hippocampal CA1 region. The
   statistical chart presents the number of BDNF/GFAP-positive cells per
   field for each group (n = 4, scale bar: 50 μm).

   (G–I) Representative western blot of C3 and BDNF proteins in the
   hippocampus. Densitometric analyses of the immunoblots were performed,
   and the results are expressed as percentages relative to the control
   group (n = 4). Con: control group; Sin lido: single lidocaine exposure;
   Rep lido: repeated lidocaine exposure. ∗p < 0.05 compared with the con
   group, ^#p < 0.05 compared with the sin lido group. Data were presented
   as the mean ± SD. See also [108]Figure S2.

   Our immunofluorescence analysis demonstrated the colocalization of
   brain-derived neurotrophic factor (BDNF) and GFAP in astrocytes within
   the hippocampus. As depicted in [109]Figures 5E and 5F, there was a
   notable decrease in BDNF/GFAP-positive astrocytes in the hippocampus
   following repeated lidocaine exposure compared to the control and
   single lidocaine groups. Additionally, western blot analysis revealed
   upregulation of C3 and downregulation of BDNF in the hippocampus of
   aged mice following repeated lidocaine exposure ([110]Figures 5G–5I).
   Taken together, the results indicate that repeated exposure to
   lidocaine promotes the transformation of astrocytes from the
   neuroprotective A2 phenotype to the neurotoxic A1 phenotype, whereas a
   single exposure does not elicit the same effect.

Repeated lidocaine exposure induced synaptic impairment and neuronal
apoptosis in the hippocampus of aged mice

   We examined synaptic morphological changes and the expression of
   synaptic markers (PSD95 and synaptophysin) using TEM and western blot
   analysis, respectively. [111]Figures 6A and 6B illustrated a
   significant reduction in the thickness of PSD in the hippocampal CA1
   area of aged mice repeatedly exposed to lidocaine compared to the
   control and single lidocaine-exposed mice. Furthermore, we observed a
   downregulation of PSD95 and synaptophysin at the protein level in the
   hippocampus of aged mice following repeated lidocaine exposure, as
   depicted in [112]Figures 6C–6E. We also investigated the levels of
   cleaved caspase-3 and Bax/Bcl-2 ratio, as they are known to play a
   crucial role in the process of cell apoptosis. Results from western
   blot analysis confirmed a significant increase in the expression of the
   apoptosis-related proteins cleaved caspase-3 and Bax following repeated
   lidocaine exposure, accompanied by a decrease in the expression of
   Bcl-2 compared to the control and single lidocaine groups
   ([113]Figures 6F–6H).

Figure 6.

   [114]Figure 6
   [115]Open in a new tab

   Repeated lidocaine exposure induced synaptic impairment and neuronal
   apoptosis in the hippocampus of aged mice

   (A and B) Representative photomicrograph (37000x) demonstrating
   variations in PSD thickness in the hippocampus. The statistical chart
   displays the PSD thickness from at least 20 sections for each group
   (n = 5, scale bar: 200 nm).

   (C–E) Representative western blot of PSD95 and synaptophysin in the
   hippocampus. Densitometric analyses of the immunoblots were performed,
   and the results are expressed as percentages relative to the control
   group (n = 4).

   (F–H) Representative western blot of Bax, Bcl-2, and cleaved caspase-3
   in the hippocampus. Densitometric analyses of the immunoblots were
   performed, and the results are expressed as percentages relative to the
   control group (n = 4). Con: control group; Sin lido: single lidocaine
   exposure; Rep lido: repeated lidocaine exposure. ∗p < 0.05 compared
   with the con group, ^#p < 0.05 compared with the sin lido group. Data
   were presented as the mean ± SD.

Pharmacological depletion of microglia attenuated A1 astrocyte activation and
synaptic impairment induced by lidocaine in the hippocampus of aged mice

   To examine the potential impact of microglial activation on astroglial
   response and synaptic function in aged mice following repeated
   lidocaine exposure, we utilized PLX5622 to deplete microglia in the
   mice’s brains. As anticipated, mice in the PLX5622 treatment group
   exhibited a marked reduction in the microglial population compared to
   mice on a control diet following lidocaine exposure ([116]Figures 7A
   and 7B). Furthermore, the levels of inflammatory cytokines, including
   IL-1α, TNF-α, and C1q, released by microglia were diminished in the
   hippocampus of aged mice after PLX5622 treatment ([117]Figures 7E–7H).
   Subsequently, the astroglial response and synaptic function were
   assessed in aged mice treated with lidocaine, with or without PLX5622.
   Following repeated lidocaine exposure, there was a noticeable increase
   in the population of A1 astrocytes and a corresponding loss of synapses
   in the hippocampus. Interestingly, depletion of microglia significantly
   ameliorated the activation of A1 astrocytes induced by lidocaine
   ([118]Figures 7C and 7D) and alleviated synaptic impairment in aged
   mice ([119]Figures 7I–7K).

Figure 7.

   [120]Figure 7
   [121]Open in a new tab

   Microglial depletion by PLX5622 treatment attenuated A1 astrocyte
   activation and synaptic impairment induced by repeated lidocaine
   exposure in the hippocampus of aged mice

   (A and B) Representative pictures of immunofluorescence staining of
   microglia (labeled by Iba-1 in green) and cell nuclei (labeled by DAPI
   in blue) in the hippocampal CA1 region. The statistical chart presents
   the number of microglia per field for each group (n = 6, scale bar:
   100 μm).

   (C and D) Representative pictures of immunofluorescence staining of
   astrocytes (labeled by GFAP in green), neurotoxic markers (labeled by
   C3 in red) and cell nuclei (labeled by DAPI in blue) in the hippocampal
   CA1 region. The statistical chart presents the number of
   C3/GFAP-positive cells per field for each group (n = 6, scale bar:
   50 μm).

   (E–H) Representative western blot of proinflammatory cytokine IL-1α,
   TNF-α, and C1qA proteins in the hippocampus. Densitometric analyses of
   the immunoblots were performed, and the results are expressed as
   percentages relative to the control group (n = 4).

   (I–K) Representative western blot of PSD95 and synaptophysin in the
   hippocampus. Densitometric analyses of the immunoblots were performed,
   and the results are expressed as percentages relative to the control
   group (n = 4). Con: control group; Lido: group that received repeated
   lidocaine exposure. ∗p < 0.05 compared with the con group, ^#p < 0.05
   compared with the lido group. Data were presented as the mean ± SD.

Inhibition of the NF-κB pathway prevented microglial activation and A1
astrocyte polarization induced by lidocaine in the hippocampus of aged mice

   To investigate the involvement of NF-κB signaling in mediating
   microglial activation and the generation of A1 neurotoxic astrocytes
   after exposure to lidocaine, we employed the specific NF-κB inhibitor
   DHMEQ. Consistent with our prior results in [122]Figure 4A, upon
   repeated lidocaine injections in aged mice, there was a significant
   increase in the number of IBA1^+ microglia in the hippocampus,
   exhibiting active cell morphology characterized by enlarged cell bodies
   and thicker processes. Nevertheless, administration of the DHMEQ
   notably reduced microglial activation, resulting in fewer IBA1^+
   microglia with a more ramified morphology ([123]Figures 8A and 8B). In
   addition, DHMEQ treatment decreased microglia-derived proinflammatory
   cytokines after repeated exposure to lidocaine, as indicated by a
   significant reduction in the expression levels of p-IκBα and p-NF-κB
   p65 ([124]Figures 8C–8E), as well as a decrease in the expression
   levels of IL-1α, TNF-α, and C1qA ([125]Figures 8F–8I). Moreover, when
   DHMEQ was administered to aged mice repeatedly exposed to lidocaine,
   the expression level of C3 significantly decreased to baseline, while
   the expression level of BDNF was remarkably rescued
   ([126]Figures 8J–8L). As expected, the RT‒qPCR results revealed that
   DHMEQ treatment significantly decreased the levels of astrocyte
   A1-specific genes (C3, H2-D1, Serping1, Amigo2) in the hippocampus of
   aged mice following repeated lidocaine exposure while increasing the
   levels of A2-specific genes (S100A10, CD14, Emp1, Stat3)
   ([127]Figure S3). These results support the idea that inhibition of the
   NF-κB signaling pathway is a plausible approach to mitigate
   neuroinflammation induced by lidocaine and prevent the neurotoxic
   polarization of astrocytes, thereby maintaining astrocytes in a
   neuroprotective phenotype.

Figure 8.

   [128]Figure 8
   [129]Open in a new tab

   DHMEQ treatment prevented microglial activation and A1 astrocyte
   polarization induced by repeated lidocaine exposure in the hippocampus
   of aged mice

   (A and B) Representative pictures of immunofluorescence staining of
   microglia (labeled by Iba-1 in green) and cell nuclei (labeled by DAPI
   in blue) in the hippocampal CA1 region. The statistical chart presents
   the number of microglia per field for each group (n = 6, scale bar:
   100 μm).

   (C–E) Representative western blot of p-IκBα, IκBα, p-NF-κB p65, and
   NF-κB p65 proteins in the hippocampus. Densitometric analyses of the
   immunoblots were performed, and the results are expressed as
   percentages relative to the control group (n = 4).

   (F–I) Representative western blot of proinflammatory cytokine IL-1α,
   TNF-α and C1qA proteins in the hippocampus. Densitometric analyses of
   the immunoblots were performed, and the results are expressed as
   percentages relative to the control group (n = 4).

   (J–L) Representative western blot of C3 and BDNF proteins in the
   hippocampus. Densitometric analyses of the immunoblots were performed,
   and the results are expressed as percentages relative to the control
   group (n = 4). Veh: vehicle group; lido: group that received repeated
   lidocaine exposure. ∗p < 0.05 compared with the veh group, ^#p < 0.05
   compared with lido group. Data were presented as the mean ± SD. See
   also [130]Figure S3.

Inhibition of the NF-κB pathway attenuated synaptic loss and neuronal
apoptosis and improved cognitive impairment induced by lidocaine in the
hippocampus of aged mice

   We next investigated whether the NF-κB signaling pathway is implicated
   in synaptic and cognitive impairment in aged mice subjected to repeated
   lidocaine exposure. DHMEQ treatment was found to reverse the
   significant reduction in PSD-95 and synaptophysin levels in the
   hippocampus of aged mice following lidocaine exposure, as shown by
   western blot analysis ([131]Figures 9A–9C). Furthermore, repeated
   lidocaine exposure resulted in an increase in levels of cleaved
   caspase-3 and the Bax/Bcl-2 ratio, which were effectively mitigated by
   DHMEQ treatment in the hippocampus ([132]Figures 9D–9F). Additionally,
   results from the MWM test demonstrated that DHMEQ treatment reduced the
   escape latency time ([133]Figures 9G and 9H), significantly increased
   the time spent in the target quadrant ([134]Figure 9I), and augmented
   the number of target crossings ([135]Figure 9J) compared to the
   repeated lidocaine group. Swim velocity was not significantly different
   among the groups ([136]Figure 9K). Taken together, these findings
   suggest that repeated exposure to lidocaine can lead to synaptic and
   cognitive impairments, primarily attributed to the activation of the
   NF-κB pathway.

Figure 9.

   [137]Figure 9
   [138]Open in a new tab

   DHMEQ treatment alleviated synaptic loss and neuronal apoptosis and
   improved cognitive impairment triggered by repeated lidocaine exposure
   in the hippocampus of aged mice

   (A–C) Representative western blot of PSD95 and synaptophysin in the
   hippocampus. Densitometric analyses of the immunoblots were performed,
   and the results are expressed as percentages relative to the control
   group (n = 4).

   (D–F) Representative western blot of Bax, Bcl-2, and cleaved caspase-3
   in the hippocampus. Densitometric analyses of the immunoblots were
   performed, and the results are expressed as percentages relative to the
   control group (n = 4).

   (G) Representative swim paths of aged mice on the fifth day of the
   place navigation and sixth day of the probe trial (n = 10).

   (H) Escape latency of aged mice to find the hidden platform during the
   5-day training trials (n = 10).

   (I) Time spent in the target quadrant during the probe test (n = 10).

   (J) Number of platform crossings of aged mice during the probe test
   (n = 10).

   (K) Swim velocity of aged mice during the 5-days training trials (n =
   10). Veh: vehicle group; lido: group that received repeated lidocaine
   exposure. ∗p < 0.05 compared with the veh group, ^#p < 0.05 compared
   with the repeated lidocaine dose-treated group. Data were presented as
   the mean ± SD.

Discussion

   Local anesthetic systemic toxicity (LAST) is a potentially
   life-threatening complication that should be carefully considered
   whenever local anesthesia is employed.[139]^32 Lidocaine remains the
   predominant local anesthetic, with a growing trend of intravenous use
   in the perioperative period. Previous animal and observational studies
   have indicated temporary cognitive impairment during convulsive
   episodes induced by the administration of local
   anesthetics.[140]^33^,[141]^34 The fact that lidocaine affects the CNS
   is not surprising, considering its ability to easily cross the
   blood‒brain barrier. Nevertheless, the molecular mechanisms underlying
   the neurotoxic effects of systemic lidocaine in CNS are poorly
   understood. A previous study employing the 2-[14C] deoxyglucose
   technique to investigate lidocaine distribution in the rodent brain
   found that lidocaine concentration is uneven, with notable accumulation
   in the hippocampus—an area recognized for its role in learning, memory,
   and cognitive function.[142]^35 Even a single application of a
   subconvulsant dose of lidocaine still resulted in damage to the
   hippocampus and amygdala neurons in adult rats, confirming that
   lidocaine can potentially cause CNS injury even at “relatively safe”
   doses.[143]^17 Given the hippocampus’s notable sensitivity to systemic
   lidocaine,[144]^17^,[145]^35 we chose to focus our study specifically
   on this brain region. We hypothesized that the effect of intravenous
   lidocaine on neurocognitive function could be influenced by the
   frequency of administration and the age of the patient. Accordingly,
   the present study aimed to evaluate the effects of different
   frequencies of intravenous lidocaine administered at a clinically
   relevant concentration on neurocognitive function across diverse age
   groups.

   In this study, the choice of dosage and administration regimen of
   lidocaine was based on the dose-conversion correlation between humans
   and mice (humans: mice = 1: 12.3).[146]^36 The objective was to
   investigate the effectiveness and safety of administering lidocaine at
   a clinically relevant concentration using an animal model.[147]^37
   Throughout the experiment, we carefully monitored the mice for any
   signs of adverse effects, such as convulsive behavior and cardiac
   toxicity. No significant signs of convulsions or hemodynamic changes
   were observed in any of the mice, even in mice with the highest
   lidocaine concentration, which remained below the previously reported
   toxic concentration following lidocaine administration.[148]^38 Our
   study demonstrated that repeated exposure to lidocaine, even at a
   clinically relevant concentration, induces cognitive impairment
   specifically in aged mice. Additionally, this impairment is correlated
   with neuronal apoptosis and synaptic loss in the hippocampus.
   Sevoflurane, an inhalation anesthetic, is widely used in surgical
   patients and has been associated with neuroinflammation and cognitive
   impairment, especially in the elderly.[149]^31 In the perioperative
   setting, the effects of repeated intravenous administrations of
   lidocaine on cognitive function following general anesthesia with
   sevoflurane were investigated. Our MWM test results further indicated
   that repeated exposure to lidocaine somewhat worsened the working
   memory impairment induced by sevoflurane in aged mice. Our findings are
   supported by the results of several human studies that have documented
   the occurrence of adverse neurological events associated with the
   administration of intravenous lidocaine.[150]^12 Andjelkovic
   et al.[151]^39 reported delirium in two patients, Dewinter
   et al.[152]^40 observed one case of tinnitus and one patient with a
   metallic taste, and Staikou et al.[153]^41 documented transient
   confusion in another patient after anesthesia recovery. Notably, none
   of the studies recorded plasma lidocaine concentrations exceeding toxic
   levels.

   The age-related response to lidocaine exposure is noteworthy. While
   aged mice exhibited cognitive impairment after repeated exposure to
   lidocaine, adult mice did not display similar deficits. Further RNA-seq
   analysis conducted on the hippocampus revealed a notable number of DEGs
   that were enriched in inflammatory responses and synaptic signaling
   after lidocaine exposure in aged mice but not in adult mice. These
   results suggest that aging may increase vulnerability to
   lidocaine-induced neurotoxicity, potentially due to age-related
   alterations in neuronal function and heightened susceptibility to
   inflammation-induced neurodegeneration.[154]^42 Additionally,
   alterations in electrophysiology associated with age intensify the
   sensitivity of the nervous system to the systemic effects of
   lidocaine.[155]^43 Although peak plasma concentrations and protein
   binding in elderly patients remain largely unchanged, the clearance of
   local anesthetics is reduced partly because of decreased organ
   perfusion and metabolic function.[156]^44 The susceptibility of elderly
   individuals to lidocaine-induced neurotoxicity emphasizes the
   significance of age as a crucial determinant when assessing the safety
   and efficacy of repetitive lidocaine administration, especially in the
   elderly population. Elucidating the mechanisms underlying this
   age-dependent response could yield insights into the varying effects of
   lidocaine on cognitive function among different age groups.

   The involvement of astrocyte polarization in mediating
   lidocaine-induced cognitive impairment is a key finding of this study.
   Repeated exposure to lidocaine resulted in the transformation
   of astrocytes from their neuroprotective A2 phenotype to a neurotoxic
   A1 phenotype in the hippocampus of aged mice. Conversely, a single
   exposure to lidocaine does not elicit the same effect. BDNF, a crucial
   neurotrophic factor predominantly secreted by A2 astrocytes, is
   essential for neuronal survival and growth, synaptic plasticity, and
   memory formation.[157]^45 In contrast, reactive A1 astrocytes have been
   demonstrated to release inflammatory mediators, including C3, leading
   to impaired neuronal viability and synaptic function. These findings
   underscore the importance of regulating astrocyte polarization and
   their interactions with neurons in mediating the neurotoxic effects of
   lidocaine.

   Anesthetics have the ability to modulate microglial activation, leading
   to both anti- and proinflammatory effects, indicating their potential
   dual roles in the development of POCD.[158]^46 Despite recent evidence
   supporting the anti-inflammatory and neuroprotective effects of
   lidocaine,[159]^47^,[160]^48 clinical studies have been unable to
   confirm its effectiveness in reducing the occurrence of POCD.
   Conversely, lidocaine has been suggested to induce neuroinflammation
   and is associated with the activation of other immune cells in the rat
   dorsal root ganglion.[161]^22 The variability among studies can be
   attributed to factors including variations in dose, timing, frequency,
   route of administration of lidocaine, and the type of tissue being
   studied. Moreover, the inflammatory environment may be influenced by
   the impact of brain background characteristics. In our study, we
   observed that repeated exposure to lidocaine induced microglial
   activation and increased the expression of proinflammatory cytokines in
   the hippocampus of aged mice. To further evaluate the influence of
   microglia on astrocytic response and synaptic function following
   lidocaine exposure, we depleted microglia in aged mice using PLX5622.
   Our study revealed that pharmacological depletion of microglia
   effectively prevented the neurotoxic activation of A1 astrocytes and
   alleviated synaptic impairment. Consistent with our findings, previous
   research has indicated that inhibiting microglia activation[162]^49 or
   eliminating microglia[163]^30 significantly improves synaptic function,
   thereby underscoring the critical role of microglial activation and
   neuroinflammation in the development of POCD. Considering the
   complexity of the pro-inflammatory effects of lidocaine, further
   research is necessary.

   The NF-κB signaling pathway, which is responsible for gene
   transcription, modulates the expression of inflammatory cytokines and
   plays a pivotal role in the regulation of inflammation and the immune
   response.[164]^50 Accumulating studies have demonstrated the
   involvement of the NF-κB pathway in mediating microglial activation and
   synaptic and cognitive impairment in AD models.[165]^51^,[166]^52
   However, there is currently no research examining the activation of
   NF-κB in relation to cognitive dysfunction associated with repeated
   exposure to lidocaine. In our study, we discovered that the NF-κB
   pathway and microglia were activated in the hippocampus of aged mice
   repeatedly exposed to lidocaine. Moreover, posttreatment with DHMEQ
   effectively inhibited the activation of the microglia and promoted the
   transition of astrocytes from the A1 to A2 phenotype. Neuronal
   apoptosis and synapse loss are reported to occur in the hippocampus
   after anesthesia and surgery,[167]^49^,[168]^53 aligning with our
   pathological observation. Furthermore, we found that inhibiting NF-κB
   activation attenuated synaptic loss and neuronal apoptosis and improved
   cognitive impairment in aged mice following lidocaine exposure.
   Overall, our findings establish the involvement of the NF-κB pathway in
   the development of cognitive dysfunction in aged mice subjected to
   repeated lidocaine exposure and that treatment with an NF-κB inhibitor
   results in substantial improvements in cognition and mitigates
   hippocampal pathology.

   In conclusion, the findings of this study provide compelling evidence
   that both age and exposure frequency are positively correlated with the
   development of cognitive impairment induced by systemic lidocaine, even
   when administered at a clinically relevant concentration. Moreover, the
   molecular mechanism underlying synaptic and cognitive impairment in
   aged mice involves the activation of microglia and neurotoxic A1
   astrocytes through the NF-κB signaling pathway. In light of the
   necessity for certain elderly individuals to undergo multiple surgeries
   as a preventive measure against recurring illnesses, it is essential to
   conduct a comprehensive evaluation of the potential benefits and risks
   linked to repeated intravenous lidocaine.

Limitations of the study

   Several limitations of this study should be acknowledged. First, the
   study primarily focused on cognitive impairments assessed by the MWM
   test. However, it is important to explore other cognitive domains,
   including attention, executive function, and anxiety behavior, to gain
   a comprehensive understanding of the effects of lidocaine on cognitive
   function. Second, the study focused on the hippocampus, and other brain
   regions, such as the prefrontal cortex or the amygdala, could also be
   affected by lidocaine exposure and should be considered in future
   investigations. Furthermore, it is crucial to explore various dosing
   intervals and investigate the long-term effects of lidocaine exposure
   to advance our understanding and clinical management of cognitive
   impairments induced by lidocaine.

Resource availability

Lead contact

   Further information and requests for resources and reagents should be
   directed to and will be fulfilled by the Lead Contact, Xiaochun Zheng
   (zhengxiaochun@fjsl.com.cn).

Materials availability

   This study did not generate new unique reagents.

Data and code availability

     * •
       The raw RNA-seq data have been deposited at NCBI and are publicly
       available. Accession number is listed in the [169]key resources
       table. The data presented in this paper will be shared by the
       [170]lead contact upon request.
     * •
       This paper does not include the original code.
     * •
       Any further information needed to reanalyze the data can be
       obtained from the [171]lead contact upon request.

Acknowledgments