See Article, p 1138
- Question: Does anesthesia accelerate the onset of Parkinson disease symptoms and pathology in an individual at risk for Parkinson disease?
- Findings: Isoflurane transiently enhanced dopaminergic neurodegeneration in the substantia nigra without inducing detectable long-term functional deficits.
- Meaning: In this animal model, isoflurane did not accelerate the onset of Parkinson disease.
Parkinson disease (PD) is the second most common neurodegenerative disorder, preceded only by Alzheimer disease. Anesthetics have been associated with the acceleration of neurodegenerative disorders in preclinical models, including Alzheimer disease1–9 and Huntington disease.10 Despite similar biophysical features of the underlying proteinopathy, less is known about an association of anesthetics with PD. This is important especially among older individuals, who are also by far the most likely to be exposed to general anesthetics and surgery. A case report described the onset of frank Parkinsonian symptoms in a 60-year-old patient immediately after surgery, which resolved during the postoperative period.11 However, 1 year later, the same patient was diagnosed with PD, suggesting that the perioperative period either transiently unmasked PD symptoms or actually accelerated PD pathogenesis. In addition, a study reported a significant increase in mortality due to PD in anesthesiologists compared to internists.12 In that study, the anesthesiologists would have been practicing in less well-scavenged operating rooms than today. Consistent with this is a case report of a 59-year-old anesthesiologist who developed PD, with evidence of poor anesthetic gas scavenging at his hospital.13 A knockout (KO) rat model of PD (DJ-1 protein, encoded by the Park7 gene [DJ-1; PARK7]), developed initially by SAGE Laboratories (now Horizon Discovery), has been shown to develop symptoms and neuropathology of PD by 8 months of age,14–16 including motor deficits and loss of dopaminergic neurons, with male rats developing symptoms more significantly than females. The DJ-1 model does not form alpha-synuclein aggregates. In this study, we hypothesized that repeated exposure to inhaled anesthetics in a presymptomatic rat model of PD would accelerate the progression of disease. We tested this hypothesis by exposing presymptomatic 6-month-old DJ-1 rats to isoflurane 3 times and then measuring the progression of neuropathology and symptoms acutely and up to 12 months of age.
Detailed methods can be found in the Supplemental Digital Content 1, Materials and Methods, https://links.lww.com/AA/D609.
The study was approved by the University of Pennsylvania Animal Care and Use Committee. All animals were treated in strict accordance with National Institutes of Health (NIH) and institutional guidelines, the United States Public Health Service Policy on Humane Care and Use of Laboratory Animals, and the Guide for the Care and Use of Laboratory Animals.17 The Animal Research: Reporting of In Vivo Experiments guidelines18 were consulted when designing the experiments. Forty-eight male homozygous DJ-1 (−/−) (PARK7) KO rats, a previously characterized model of PD14 developed from Long Evans hooded rats, were obtained at 8 weeks of age from SAGE Laboratories (Boyertown, now Horizon Discovery). Males show greater PD progression than females in this model.14
Anesthetic Exposures and Monitoring
All rats were 6 months old at the time of the first anesthetic exposures. The exposed animals (maximum 9 rats at 1 time) were placed in a large (27” × 14” × 12”) gas-tight acrylic chamber (Cleatech). The chamber was equipped with gloves so rats could be manipulated and monitored without opening the chamber. Isoflurane (2% in 30% oxygen balanced with nitrogen for induction, followed by 1.2% isoflurane for maintenance) flowed into the vented chamber. A gas analyzer monitored the isoflurane, oxygen, and carbon dioxide concentrations. Heart rate, respiratory rate, oxygen saturation (Spo2; PhysioSuite and rat paw sensor, Kent Scientific), and rectal temperature were recorded every 10 to 15 minutes on select rats. The rats were anesthetized for 2 hours, and anesthesia was repeated at 2 and 4 weeks later. If any rat showed signs of pain or distress, including significant loss of body weight (body conditioning scores [BC] of BC1 or BC2 with other signs including piloerection, rough/stained haircoat, abnormal arched back, porphyrin staining), it would be immediately euthanized. Body weight for all groups from 6 months of age until euthanasia was recorded (Supplemental Digital Content 2, Figure S1, https://links.lww.com/AA/D610).
Motor function and memory were assessed using the ladder rung test, rotarod, and object recognition, as shown in the timeline (Figure 1). Our primary outcome for this study was coordinated motor behavior. The testing regimen was different for the acute group (AG) and the long-term group (LTG), on the basis of our hypotheses. For the AG, our hypothesis was that the animals’ motor behavior would acutely change within days after the exposure, and that cumulative exposures would worsen the progression, as has been reported in wild-type and transgenic Alzheimer mouse models. We tested the AG after each of the 3 exposures, 2 weeks apart. In addition, we tested right before and right after the third exposure to examine the before-and-after scenario. The hypothesis for the LTG was that after the same 3 exposures, the progression of PD would be accelerated with age. The AG underwent ladder rung (1 day after each isoflurane exposure) and rotarod (2 days after each exposure) testing, ending at 7 months of age. In addition, the AG underwent the same motor tests 1 and 2 days before the third exposure. The LTG began the same testing regimen 4 weeks after the third exposure and then again at 3- to 4-week intervals until they were 12 months of age. Cognitive dysfunction was not a primary outcome for this study as cognitive deficits are not early or pronounced in patients with PD, especially in midlife, and cognitive dysfunction has not been reported in this model. We decided to evaluate a subset of animals in the LTG with the novel object recognition (NOR) test as a secondary end point to determine whether 12-month-old PD rats exhibited cognitive decline.
Horizontal Ladder Rung Task
The Horizontal Ladder Rung Test19 was used to assess motor function and coordination in the rats. After all animals completed the training, the test was repeated for 4 test trial runs, each with a different predetermined rung pattern (Figure 2E). The slow-motion video recordings were analyzed after the rats were euthanized, and the total number of foot faults were manually recorded for each of the 4 rung patterns. The data were analyzed for both the total number of foot faults for each rung pattern and the total number of foot faults per animal. This procedure was performed at 4 time points for the AG and 6 time points for the LTG.
The Accelerating Rotarod Test20 was used to assess motor coordination and balance. The time spent on the rod was recorded for each animal. Animals were tested at 4 time points for the AG and 6 time points for the LTG. However, unexpectedly, some of the rats, irrespective of experimental group, refused to perform all 3 testing trials at some of the time points. The mean time spent on the rotarod at each time period was recorded for the analyses.
Novel Object Recognition
The NOR test21 was performed at 12 months of age in a subset of rats to assess cognitive function, specifically short- and long-term memory, and rearing. The rats were video-recorded and tracked using the AnyMaze software to determine the time spent exploring each object, as well as the time spent rearing.
All rats were euthanized after their respective behavioral testing, at 7 months of age for the AG and 12 months of age for the LTG. Terminal surgery was performed under 4% to 5% isoflurane anesthesia balanced with 30% oxygen and nitrogen, by left ventricular intracardiac perfusion with ice cold phosphate-buffered saline and concomitant atrial exsanguination.
After euthanasia, the dissected brains were cut, fixed in 4% paraformaldehyde (Fisher Scientific), and processed for paraffin embedding. Rat brains from the AG and LTG were randomly chosen to be sectioned for immunohistochemistry. Due to technical issues with sectioning, the animal numbers for the AG were n = 5 controls, n = 6 exposed, and for the LTG n = 11 controls and n = 9 exposed. Slides were quantified by the same 2 blinded investigators, and the means of their results were used for each animal.
The primary outcome was the ladder rung test, and secondary outcomes were the other behavioral and immunohistochemical assays. For the AG ladder rung and rotarod tests, sample size was determined using an expected large effect size f of 0.7 (due to acute nature of the AG), alpha error probability of .05, power of 0.80, 2 groups, 3 time points, correlation among repeated measures of 0.5, and nonsphericity of 1; the sample size was 6. For the LTG, using an expected medium effect size f of 0.3 (due to the long-term nature), alpha error probability of .05, power of 0.80, 2 groups, 6 time points, correlation among repeated measures of 0.5, and nonsphericity of 1, the sample size was 14. Because the DJ-1 model was newly developed with no data on mortality at 12 months of age, we allowed for possible illness or mortality by 12 months and increased sample size to n = 18. For the ladder rung test and rotarod data analyses for the AG and LTG, we used repeated measures 2-way analysis of variance (ANOVA) and/or 3-way ANOVA. For the NOR test and the immunohistochemical assays, the sample size was determined based on the literature22,23 and from our laboratory,24–26 and an unpaired t test with Welch’s correction was used for the analyses, with the data tested for normality (Shapiro-Wilks test). Sample sizes are presented in the Results and in figure legends for each analysis. The statistical analyses were performed with unblinded data using GraphPad Prism v8.4 - 9.0 for Windows (GraphPad Software, www.graphpad.com) or R, v 4.0.2, (https://www.r-project.org/), and are described in detail in the Results for each analysis. For all data, α = .05 and significance was determined to be P < .05 (2-tailed); data are presented as the mean with 95% confidence intervals (CIs). Data are available in the Harvard Dataverse online data repository (https://dataverse.harvard.edu/dataset.xhtml?persistentId=doi:10.7910/DVN/MKDXMP).
The isoflurane-exposed and control DJ-1 rats underwent behavioral testing to assess motor and cognitive function (Figure 1).
To evaluate motor function, the AG underwent training on the ladder rung and rotarod immediately after each of the 3 isoflurane exposures to evaluate behavior with multiple exposures and before the third isoflurane exposure to examine behavior immediately before and after an exposure.
AG Ladder Rung
The rats had no difficulty navigating any of the rung patterns on the ladder rung test.
AG Total Number of Foot Faults
No significant differences were detected in the total number of foot faults with the repeated measures 2-way ANOVA with Geisser-Greenhouse correction, with variables of time and exposure, P = .10, F(2, 20) = 2.55, and with Sidak’s multiple comparisons tests between the control and the isoflurane-exposed groups after each of the 3 exposures (adjusted P values: P = .64, P = .97, P = .80, respectively; Figure 2A). However, both controls and exposed groups performed significantly better (decreased number of foot faults) from the first exposure to the third exposure (adjusted P values, P = .05 and P < .001, respectively) and from the second to the third exposure (adjusted P values, P = .01, P = .002, respectively; n = 6 rats per group; Figure 2A). In addition, no significant differences were detected in the AG tested immediately before and after the third exposure with the repeated measures 2-way ANOVA with Geisser-Greenhouse correction (time × exposure, P = .45, F(1, 10) = 0.63) and with Sidak’s multiple comparisons tests between the controls and exposure groups before and after the third exposure (adjusted P values: P = .12, P = .56, respectively; n = 6 rats per group; Figure 2B). There was also no difference in the control group before and after the exposures (adjusted P = .11) or in the isoflurane group (0.56).
AG Rung Patterns
No significant 3-way interactions between exposure, time point, and ladder rung pattern were detected for the AG, P = .71, F(6, 60) = 0.63, with the repeated measures 3-way ANOVA with Geisser-Greenhouse correction using a type III sum-of-squares (n = 6 rats per group; Figure 2C). On the other hand, 2-way interactions between exposure and ladder rung pattern, P = .02, F(3, 30) = 3.89, exposure and time point, P = .01, F(2, 20) = 5.60, and ladder rung pattern and time point, P = .02, F(6, 60) = 2.79, were all significant. However, when the effect of exposure was tested at each level of time point and ladder rung pattern, no significant P values were reported using Holm’s multiple comparisons test. By contrast, when the effect of time point was tested at each level of exposure and ladder rung pattern, the effect of time point was found to be significant for exposed animals on ladder rung pattern 1 (Figure 2C, first panel) using Holm’s multiple comparisons test (adjusted P = .04). Specifically, using Holm’s multiple comparisons test to perform post hoc simple–simple comparisons, we found that the exposed group performed significantly better (decreased number of foot faults) on ladder rung pattern 1 from the first exposure to the third exposure (adjusted P = .03) and from the second exposure to the third exposure (adjusted P = .01; Figure 2C, first panel).
For the “before and after the third exposure” analysis (Figure 2D), we used 3-way repeated measures ANOVA (exposure, time point, and ladder rung pattern) with Geisser-Greenhouse correction using a type III sum-of-squares (n = 6 rats per group) and observed a significant effect for the 3-way interaction, P = .05, F(3, 30) = 2.98. While further analysis revealed that none of the simple 2-way interactions were significant, the effect of ladder rung pattern specifically in control animals was found to be significant using Holm’s method to adjust for multiple comparisons, adjusted P = .01, F(3, 15) = 8.53. More specifically, examination of the simple–simple comparisons with Holm’s multiple comparisons correction showed that this could be attributable to control rats performing significantly better than exposed rats after the third exposure, specifically on ladder rung pattern 2 (adjusted P = .04; Figure 2D, second panel). The 4 ladder rung patterns are depicted in Figure 2E.
Due to inconsistency in animal performance on the rotarod task, as described in the Methods, no data were available for analysis after the first and second exposures for the AG. Rotarod performance before and after the third exposure was analyzed with repeated measures 2-way ANOVA, which detected no significant differences between the groups (time × exposure), P = .35, F(1,8) = 1.01. With Sidak’s multiple comparison tests, no differences were found between control and exposure groups (adjusted P values: P = .87 and P = .19, respectively) or within groups (adjusted P values: P = .79 and P = .20, respectively), before and after the third exposure (n = 6 controls, n = 4 exposed; Figure 2C).
The rats in the LTG were initially trained on the ladder rung and rotarod at 8 months of age (4 weeks after the third exposure) and then tested regularly (every 3–4 weeks) until 12 months of age.
LTG Total Number of Foot Faults
As with the AG, the rats in the LTG had no difficulty performing the ladder rung test. No significant differences were detected in the total number of foot faults (P = .15, n = 18 controls and n = 16 exposed rats) with repeated measures 2-way ANOVA with Geisser-Greenhouse correction with variables of time and exposure, P = .15, F(5, 160) = 1.66, nor between controls and isoflurane-exposed rats at each time point with Sidak’s multiple comparisons test (adjusted P values for the 6 time points, respectively, P > .99, P = .51, P = .41, P = .73, P > .99, P > .99; Figure 3A).
LTG Rung Patterns
For the ladder rung test, no significant 3-way interactions between exposure, time point, and ladder rung pattern were detected, P = .60, F(6.83, 116.08) = 0.78, with the repeated measures 3-way ANOVA with Geisser-Greenhouse correction using a type III sum-of-squares (n = 8 control animals, n = 11 exposed animals; Figure 3B). Video technicalities resulted in 10 controls and 5 exposed rats having to be omitted due to lack of complete data at each time point for the ladder rung pattern analyses. Moreover, none of the effects involving exposure were found to be significant, including the 2-way interaction between exposure and time point, P = .09, F(5, 85) = 1.99, the 2-way interaction between exposure and ladder rung pattern, P = .09, F(2.04, 34.64) = 2.59, and the main effect of exposure, P = .68, F(1, 17) = .172. However, the 2-way interaction between time point and ladder rung pattern was found to be significant, P = .03, F(6.83, 116.08) = 2.36; when further investigated, we found that when using Holm’s method to adjust for multiple comparisons, the effect of time point was significant for control animals on ladder rung pattern 1, adjusted P = .02, F(5, 35) = 4.57.
As with the AG, the LTG rats also frequently refused to perform the accelerated rotarod test. For the analyses, the best 2 of 3 trials were used for the repeated measures 2-way ANOVA with Geisser-Greenhouse correction. The first time point for the rotarod testing had to be omitted due to incomplete data, and for the remaining time points, 5 controls and 2 exposed rats had to be omitted due to lack of complete data at each time point (n = 13 controls, n = 14 exposed). No significant difference was detected in the repeated measure 2-way ANOVA (time × exposure), P = .08, F(4, 100) = 2.18, and with Sidak’s multiple comparisons test, there were no significant differences between the controls and exposed groups at any time point (adjusted P values, P = .69, P = .99, P = .99, P = .99, P = .98, time points 2–6, respectively; Figure 3C).
Novel Object Recognition
As a secondary end point, cognitive function in a subset of animals was examined at 12 months of age to test the hypothesis that anesthetics accelerated the onset of cognitive decline in PD-vulnerable rats. The NOR test was used to assess cognitive function (n = 8 controls, n = 9 exposed; technical error with tracking software resulted in no data for 1 control rat). There were no differences in short-term memory (P = .57; Figure 3D), long-term memory (P = .47; Figure 3E), or in the number of rearings (P = .10; Figure 3F) between controls and isoflurane-exposed rats using the unpaired t test with Welch’s correction.
Following their respective behavioral assays, the AG and LTG were euthanized for immunohistochemical assays (tyrosine hydroxylase [TH] and ionized calcium-binding adaptor protein-1 [Iba-1]) at 7 and 12 months of age, respectively.
Brain sections through the substantia nigra pars compacta (SNpc) were stained for TH from the AG and LTG groups (Figure 4A–D). The AG was found to have a significantly lower density of TH-containing cells in the SNpc (P = .04; n = 5 controls, n = 6 exposed) compared to controls using an unpaired t test with Welch’s correction (Figure 4E) while, for the LTG, no significant difference was detected in the density of these neurons (P = .24; n = 11 controls, n = 9 exposed; Figure 4F).
Ionized Calcium-Binding Adaptor Protein-1
The hippocampal CA1 region (Figure 5A–D) and substantia nigra (Figure 6A–D) were examined for microglial activation, measured by the density of Iba-1–positive microglial cells, in the AG and LTGs. No significant differences between isoflurane-exposed and controls were detected in the density of microglial cells in the CA1 region in the AG or the LTG (P = .11 [n = 5 controls, n = 6 exposed], P = .54 [n = 14 controls, n = 12 exposed], respectively; Figure 5E, F). Likewise, the density of Iba-1–positive microglia in the SNpc showed no significant differences in the AGs (P = .82, n = 3 controls, n = 4 exposed) or LTGs (P = .38, n = 4 controls, n = 3 exposed; Figure 6E, F).
Anesthetic acceleration of amyloidopathy and tauopathy in preclinical animal models has been well documented, although less well documented in clinical studies. PD is another common proteinopathy, for which only limited clinical evidence of anesthetic modulation exists. Nevertheless, transient movement disorders resembling PD are fairly common during recovery from general anesthesia, and no studies have searched for an association between incident PD and prior anesthesia/surgery. Thus, we undertook this study with the hypothesis that isoflurane anesthesia alone would accelerate the neuropathology and symptomatology associated with an existing PD vulnerability. The DJ-1 rat is a PARK7 KO model and has been reported to develop dopaminergic neuron loss, as well as progressive movement disorders resembling PD compared to wild-type rats, with 50% of rats being affected by 8 months of age.14–16 This model therefore should allow the detection of either acceleration or deceleration of the disease process.
The ladder rung test was sufficiently sensitive to detect a learning effect in both groups over 6 weeks of testing; thus, our inability to detect an effect of the isoflurane exposure is unlikely to have been due to the use of an insufficiently sensitive assay. Similarly, there were no differences in weight gain (Supplemental Digital Content 2, Figure S1, https://links.lww.com/AA/D610), grooming, or other general features of daily living, all dependent to a degree on mobility and coordination. Despite this, we detected a significant decrease in TH-containing cells in the SNpc in the acute-exposed group. That the effect was not dramatic enough to elicit motor or behavioral changes is consistent with previous research suggesting that the much larger inflammatory insult of a surgical procedure is necessary to produce functional changes. Other possible explanations are that the animal either was robust enough to compensate for small decreases in dopaminergic signaling or had sufficient dopaminergic reserve to not produce a phenotype. Interestingly, and also consistent with our previous study,26 the effects caused by the anesthetic alone resolved as the animal aged to 12 months.
The mechanism(s) responsible for the transient change in TH expression is not clear. It may indicate an activation of neuroinflammation, although significant microglial activation was not found in either the hippocampus or substantia nigra. We and others have reported no increase in neuroinflammation after anesthesia alone,26,27 so this is not unexpected. Alternatively, the decrease in TH expression might be a direct effect of isoflurane alteration in dopaminergic signaling pathways,28 or a compensatory change in expression due to an anesthetic-induced increase in TH activity.29 Leikas et al,28 however, reported no changes in TH protein shortly after several brief exposures to isoflurane in a mouse model of PD, with improved sensorimotor function. Regardless, the transient acute decrease in TH expression is likely of little significance as it does not appear to be persistent or progressive, and therefore is unlikely to reflect an anesthetic acceleration of PD neuropathology.
There are several limitations and future directions from this study. Characterizing subtle motor defects in a quadruped can be challenging, and there are assays available other than those we used. However, the ladder rung task was sensitive enough to detect learning in the 2 groups, and therefore we believe should have detected a functionally relevant change due to the anesthetic, if present. Also, rats cannot always be successfully encouraged to complete a task, as we found in the rotarod task and noted elsewhere.30 Thus, our rotarod data are missing a considerable number of animals and should therefore be considered to have significant selection bias, although we do not know whether refusal to perform the rotarod indicates high or low performance. The transient decrease in the density of dopaminergic neurons may not be real and could be due to type I error, especially since the density was calculated as the number per area and was not performed using stereological methods. Another limitation is that only male DJ-1 rats were used in this study, based on previous research indicating that male rats develop symptoms more significantly than females.14 Finally, anesthesia is rarely given in the absence of surgery or some other painful and inflammation-provoking procedure. We found previously that the addition of a simple surgical procedure converted a transient memory defect in mice to a much more durable one26; thus, it is possible that the addition of surgery here could have done the same.24,26 This has also been proposed for studying postoperative neurocognitive disorders in orthopedic patients at risk for PD.31
In summary, repeated exposures to isoflurane in the prodromal phase of PD transiently accelerates the loss of TH expression in a rat model of PD, in the absence of an acute motor effect and with no long-term detrimental effects on motor skills or neuropathology. We conclude that in this animal model, isoflurane alone does not accelerate the onset of PD.
Name: Daniel A. Xu, BS.
Contribution: This author helped perform experiments and collect data, conduct statistical analyses, prepare figures, and write and edit the manuscript.
Name: Timothy P. DeYoung, MSN, RN.
Contribution: This author helped conceive of and design the experiments, perform experiments and collect data, analyze data, and write and edit the manuscript.
Name: Nicholas P. Kondoleon, MD.
Contribution: This author helped perform experiments and collect data, prepare figures, and write and edit the manuscript.
Name: Roderic G. Eckenhoff, MD.
Contribution: This author helped conceive of and design the experiments and edit the manuscript.
Name: Maryellen F. Eckenhoff, PhD.
Contribution: This author helped conceive of and design the experiments, perform experiments and collect data, conduct statistical analyses, prepare figures, and write and edit the manuscript.
This manuscript was handled by: Oluwaseun Johnson-Akeju, MD, MMSc.
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