Increased sensitivity in detection of deficits following two commonly used animal models of stroke

Abstract

Stroke is a leading cause of death and disability in the United States. Most strokes are ischemic, resulting in both cognitive and motor impairments. Animal models of ischemic stroke such as the distal middle cerebral artery occlusion (dMCAO) and photothrombotic stroke (PTS) procedures have become invaluable tools, with their own advantages and disadvantages. The dMCAO model is clinically relevant as it occludes the artery most affected in humans, but yields variability in the infarct location as well as the behavioral and cognitive phenotypes disrupted. The PTS model has the advantage of allowing for targeted location of infarct, but is less clinically relevant. The present study evaluates phenotype disruption over time in mice subjected to either dMCAO, PTS, or a sham surgery. Post-surgery, animals were tested over 28 days on standard motor tasks (grid walk, cylinder, tapered beam, and rotating beam), as well as a novel odor-based operant task; the 5:1 Odor Discrimination Task (ODT). Results demonstrate a significantly greater disturbance of motor control with PTS as compared with Sham and dMCAO. Disruption of the PTS group was detected up to 28 days post-stroke on the grid walk, and up to 7 days on the rotating and tapered beam tasks. PTS also led to significant short-term disruption of ODT performance (1-day post-surgery), exclusively in males, which appeared to be driven by motoric disruption of the lick response. Together, this data provides critical insights into the selection and optimization of animal models for ischemic stroke research. Notably, the PTS procedure was best suited for producing disruptions of motor behavior that can be detected with common behavioral assays and are relatively enduring, as is observed in human stroke.

In the United States, approximately 795,000 people will experience a stroke each year, 87% of which are ischemic [35]. Treatments for ischemic stroke are aimed at reducing tissue loss by returning blood flow to the affected area. Recombinant tissue plasminogen activator and other endovascular therapies are standard treatments to break up the blood clot [38]. Given the limited window of effectiveness for these treatments, alternative methods for lessening tissue loss after ischemic stroke are needed. Development of novel therapeutic options would greatly benefit from refinement of current animal models of ischemia and how they are assessed.

Two of the most utilized procedures for preclinical mouse models of ischemic stroke are distal middle cerebral artery occlusion (dMCAO) and photothrombotic stroke (PTS) induction. While both induce permanent focal ischemia in the cortex, each procedure has distinct advantages and disadvantages. By occluding the middle cerebral artery, the most common artery affected in ischemic stroke, the dMCAO model is extremely clinically relevant. The neuroanatomical location of the middle cerebral artery varies between subjects, yielding variability in the location of infarct. However, dMCAO produces consistent lesion sizes, a clear infarct border, and low mortality rate, all of which significantly reduces experimental variability [10,20,24]. The consistent lesion size and infarct border produced in dMCAO is beneficial for examining the inflammatory response and infarct expansion. Yet, in some mouse strains, the lesion size can be minimal and may not extend into the motor cortex [10]. Induction of dMCAO is also difficult and time consuming to learn and perform due to the mortality risk associated with the craniectomy.

Alternatively, the PTS model involves administering a photoreactive dye and placing a light source over a brain structure of interest [2,20]. For example, the motor cortex can be targeted to induce reliable fine and gross motor deficits that can be measured following experimental manipulations. Impairments commonly examined with PTS model include deficits in locomotor function, sensorimotor function, and short-term memory [9,11,14,27]. While the PTS model has been shown to produce consistent infarct size and location, it may be less clinically relevant. The process by which stroke is induced with the PTS model does not occur naturally and results in thrombosis of all vasculature within the targeted area [8].

While both models can typically be incorporated into an experimental paradigm, understanding the behavioral and clinical relevance of each model is crucial. The dMCAO model targets the most affected artery in human ischemic stroke and is a better model for examining infarct and inflammatory changes between groups. However, without behavioral tasks sensitive enough to measure the deficits produced by this procedure, the model is not useful for evaluating functional recovery. While PTS leads to easily detectable deficits, it is unclear how well PTS reflects the processes involved in human stroke recovery. Identifying an animal model with both clinical relevance and easily measurable functional deficits would be beneficial. Regarding task selection, cross-species tasks utilizing analogous procedures for human participants and model species would be particularly useful for confirming the translational merits of stroke induction procedures.

The five-choice continuous performance test (5C-CPT) is a cross-species task of sustained attention offering unique features that are potentially useful for evaluating the effects of stroke induction [21,40,41]. The task extends from the five-choice serial reaction time task (5CSRTT) that has been used extensively to characterize attention and impulsivity [3,31]. The 5C-CPT involves “target” and “non-target” trials in which the subject responds to target trials and inhibits responses on non-target trials. The 5C-CPT measures attention, impulsivity, motivation, and motor impairment simultaneously and separately, which is ideal to determine which abilities are contributing to performance. A unique aspect of the 5C-CPT task is that testing procedures and performance interpretation are designed for cross-species use, with analogous procedures developed for rodents and humans [41].

A novel olfactory version of the 5C-CPT has been developed, the 5:1 Odor Discrimination Task (ODT). For the ODT, an olfactometer presents five target odors and one non-target odor. Target trials present a single target odorant and responses, in the form of licking on a sensor, are rewarded with sucrose solution. During non-target trials, the non-target is presented, with responses punished by a time-out. With the incorporation of both target and non-target trials, signal detection metrics can be computed that separately reflect composite accuracy (sensitivity index) and response/non-response bias (responsivity index).

The visual 5C-CPT can require several months of training for mice to achieve accurate and stable performance [16,28]. However, the use of olfactory stimuli greatly reduces training time in comparison to visual tasks in rodents. Several aspects limit visual abilities in mice. In contrast with humans, mice possess a more restricted visual field and have less wavelength-sensitive photoreceptors in their retinas than humans [17,22]. Variability in visual acuity between strains of mice has shown to account for inter-strain variability in visuospatial learning tasks such as the Morris water maze [5]. Though the rodents used in experimental investigation have comparatively poor vision, they are known to have an excellent sense of smell, serving as a primary sense in these species [1,36]. Accordingly, operant training using olfactory stimuli is often accomplished more rapidly and can yield higher levels of performance as compared to visual or visuo-spatial stimuli.

The present study trained mice to perform an odor-based variant of the 5C-CPT, the ODT, and evaluated performance after Sham surgery, dMCAO, or PTS induction. The use of the ODT allowed for concurrent evaluation of performance (i.e., accuracy) and motivation (i.e., responsivity). Additionally, via use of the lick sensor, licking behavior over the response period could be well characterized. As a result, both cognitive deficits (e.g., reduced accuracy) and motor impairments (e.g., altered lick patterns) can be detected concurrently. It was expected that licking behavior would be sensitive to motoric disruption induced by stroke [39].

Research on humans who have experienced a stroke demonstrates that olfactory impairment can occur if blood flow is lost to areas of the brain responsible for olfactory processing, such as impairment to anterior cerebral circulation and/or posterior circulation which affects blood flow to the insular, parietal, and orbitofrontal cortices [12,13]. The impairment that is most often observed is an inability to identify odorants, or assign verbal labels to a smell [37]. The pure ability to detect an odorant, olfactory sensitivity, is not typically affected following ischemic stroke [26]. Though olfactory sensitivity impairment is rarely reported following ischemic stroke, cases of impairment most often result from a middle cerebral artery occlusion due to loss of blood flow to the insular frontoparietal areas [23]. Of all stroke types, only about 10% of individuals experience deficits in olfactory ability following a stroke [13]. Therefore, it is not expected that deficits on the ODT will result from impairments of the ability to detect odorants, particularly due to rodent’s highly sensitive olfactory abilities.

To develop future therapeutics for stroke, animal models must be developed with a mechanism, pathophysiology, and behavioral response similar to humans. This is the first utilization of the ODT task to measure cognitive and motor performance following any animal model of ischemia. Additionally, performance on motoric behavioral tasks (i.e., cylinder task, grid walk, tapered beam, and rotating beam) were compared with performance on the novel ODT task following two models of stroke. We were able to determine the sensitivity of the ODT in detecting motor impairments while also determining if attention and motoric impulsivity are disrupted by stroke. The use of two stroke models also allowed for direct comparison of subsequent motor and/or cognitive effects, longitudinally through the acute, sub-acute, and chronic phases of recovery.

1. Materials and methods

1.1. Subjects and design

Forty-five male and female C57BL/6J mice obtained from Jackson Laboratory (Ellsworth, ME) began training between 12 and 17 weeks of age (22 females, 23 males). Mice were housed in groups of two to four in a temperature and humidity-controlled vivarium with a reverse light cycle. All subjects were maintained at 85–90% of their free-feeding weight and had ad libitum access to water, except during training and testing. Mice wererandomly assigned mice into three groups: dMCAO (n = 17), PTS (n = 13), and sham (n = 16). All mice underwent pretraining and baseline assessment with motor and cognitive behavioral assessments (see Fig. 1A). All procedures were approved by the University of North Carolina Wilmington Institutional Animal Care and Use Committee.

Fig. 1.

Photothrombotic stroke had worsened functional recovery after ischemic stroke. (A) Our experimental timeline integrated cognitive and motor assessments in two models of ischemic stroke. The ODT comprised three training phases: head entry, sustained head holding, and 5:1 (Go: No Go) discrimination task. Motor assessments included the Cylinder Task, Grid Walk, Tapered Beam, and Rotating Beam, to assess fine and gross functional recovery. Created with BioRender.com. (B) The PTS mice were significantly more reliant on the uninjured forelimb compared to Sham and dMCAO mice in the cylinder asymmetry task (F = 6.2, p =.0001). A post-hoc analysis revealed PTS mice were more reliant on the uninjured limb only on 3 DPI (p <.05). (C) PTS mice made significantly more foot faults on the contralateral limb compared to Sham mice on the grid-walk task (F = 6.2, p =.0001). A post-hoc analysis revealed PTS mice made more errors 1- (p <.05), 3- (p <.001), 7-, 14-, and 28 DPI (p <.01). There was no difference between dMCAO and Sham at any of the time points. (D) PTS mice made significantly more contralateral limb errors compared to Sham mice (F = 15.5, p =.0001). A post-hoc analysis revealed PTS mice made more errors 1-, 3- (p <.0001), and 7 DPI (p <.05). There was no difference between dMCAO and Sham at any of the time points. (E) PTS mice traveled a significantly shorter distance until falling from the rotating beam compared to dMCAO and Sham mice (F = 24.9, p =.0001). A post-hoc analysis revealed PTS mice fell at shorter distances from the beam 1-, 3-, and 7 DPI (p <.001). There was no difference between dMCAO and Sham at any of the time points. * p <.05, ** p <.01, *** p <.001 (PTS compared to Sham); # p <.05, ## p <.01, ### p <.001 (PTS compared to dMCAO). Error bars represent mean standard error. Abbreviations: ODT, odor discrimination task; PTS, Photothrombotic; dMCAO, distal middle cerebral artery occlusion. Note: Behavior and weight were not different pre-stroke among groups.

1.2. Stroke models

Mice were anesthetized using 2% Isoflurane in 2 L/min oxygen and temperature was maintained at 37 °C. For the 24 h and 7-day sacrifice timepoints, mice underwent a dMCAO to create a primary cortical infarct [10,27]. Briefly, an incision was made in the skin over the temporalis muscle to expose the middle cerebral artery through the skull. A craniotomy was performed and the medial cerebral artery was then cauterized. The temporalis muscle and skin were replaced and the incision closed. Buprenorphine (0.1 mg/kg) and Cefazolin (25 mg/kg) were subcutaneously injected.

To assess chronic inflammation and functional recovery, PTS induction was performed, targeting the motor cortex. Mice were injected (intraperitoneal) with Rose Bengal (10 mg/ml in sterile saline; 40 mg/kg of body weight), anesthetized, and then mounted on a stereotaxic frame. After incision of the scalp, and visualization of the skull, a Metal Halide Fiber optic light (no. 56371, Edmund Optics) with a 1 mm diameter contacted the skull over the right motor cortex (+.5 mm anterior, +1.5 mm lateral to bregma) for 15 mins. Sham animals received the same 40 mg/kg injection of Rose Bengal, were placed on the stereotaxic frame, and had a comparable incision, but there was no exposure to the fiber optic light. The incision was closed and a subcutaneous injection of Buprenorphine (0.1 mg/kg) and Cefazolin (25 mg/kg) was administered.

1.3. Functional recovery assessment

Motor behavior tasks were performed in dMCAO, PTS, and Sham for 28 days after surgery and included the cylinder task, grid-walk, tapered beam, and rotating beam. All behavioral tasks included three baseline measures one week prior to the stroke (one habituation day, and two pretraining days) and then assessed on motor performance 1-, 3-, 7-, 14-, and 28 days post-injury (DPI). The rotating beam was scored live by a researcher blinded to experimental conditions. All other behavioral tasks were video recorded and later analyzed by one research assistant who was blind to the experimental manipulations. Inter-rater reliability was determined on 25% of the data by a separate research assistant who was also blind to the experimental manipulations. Differences between raters’ scores for each task were determined with paired student’s t tests. There were no significant differences between raters’ scores for any of the tasks.

The cylinder task is a sensorimotor task that measures spontaneous forelimb and fine motor use. It was performed as previously described [2,32]. Briefly, each mouse was placed in a 500 ml glass beaker and recorded from above for 5 mins. As the mouse naturally reared to explore the container, the forelimb used to make contact was recorded as either “left” or “right”, and if both forelimbs were used simultaneously or with lateral movements to move along the wall, a recording of “both” was used. In the instances where one forelimb made contact first, and then the other forelimb made contact without the removal of the first forelimb, an “independent” and “both” recording were used. The results are expressed as a percentage of (ipsilateral/uninjured) forelimb used relative to the total number of contacts.

The grid walk task is used to measure gross motor impairments. Mice were placed on a raised wire grid (39 cm × 25.5 cm) with 1 cm between wires and recorded for 5 mins. The number of foot faults were scored for each forelimb and hindlimb. A foot fault was determined as any step where the foot went through a grid hole or when the animal was resting with the wrist at the same level as the grid [2,27]. The results are expressed as a percentage of (contralateral/injured) foot faults.

The tapered beam task was used to assess gross locomotor impairments. Briefly, mice were placed on the wide end of a beam that gradually tapered down from 3.3 cm to 0.5 cm across and was 100 cm long. Three trials per session were performed by each mouse. An error was determined when the mouse hind or forelimb slipped off of the beam, and the distance to the first contralateral error and of total number of contralateral errors were scored [27,32].

The rotating beam task was used to assess balance and coordination impairments. Mice performed four trials per session. A slowly rotating beam (6 rpm) measuring 120 cm was used, and mice were trained to run across the beam. The distance to fall was recorded as the primary outcome measure [7,27].

Following ODT training and baseline motor assessment, subjects received a stroke induction procedure (dMCAO stroke, PTS, or Sham surgery). Group assignment was counterbalanced by baseline ODT performance on the final day of training. Subjects were then tested for an additional 28 days (Monday-Friday) on the ODT for a total of 20 sessions. On days where ODT and other motor behavior tasks occurred, the ODT task was performed first.

1.4. Olfactometry

An olfactometer (Knosys Inc.), was used to train all subjects in the 5:1 ODT. The olfactometer utilized six odor-stimuli containers in which sterile water (100 ml) and pure extracts (6.7 ml) were mixed to produce odorants. Odors used in the current study were cinnamon, lime, peppermint, coffee, raspberry, and rum. Odor valves connected to each odorant container allowed the air flow through containers to be individually controlled. Inside the animal chamber was an opening to the sampling port, a glass tube through which air from the olfactometer can be directed and sampled upon opening of the “final valve”. The entry to the sampling port was gated by photobeams to detect head-entry and initiate trials. A lick sensor was available within the sampling port, to measure licking behavior throughout each trial (sampled at 50 Hz). A computer-operated valve allowed for the delivery of a 20% sucrose solution [weight (g)/volume (ml)] through the lick sensor as a reward.

1.4.1. Head-entry and head-hold training

Olfactometer training began with the “head-entry” phase, in which trained subjects were trained to enter the sampling port. Subjects were free to explore the chamber and a reward was delivered for each entry into the sampling port. Training continued until 30 trials of head-entry were recorded in a single session. Following head-entry training, subjects advanced to the “head-hold” phase. In this phase, an odor was released upon entry into the sampling port. This training program had six different requirements for duration of head-holding. First, subjects were required to enter the sampling port and remain for 0.1 s to initiate reward delivery. Subsequently, they were moved through additional phases of increasing durations. Training continued until subjects completed all 40 trials at the longest hold duration (1.1 s) on two consecutive sessions.

1.4.2. 5:1 Odor discrimination task

Following head-entry and head-hold training, subjects were trained on the 5:1 ODT. Six odors were used in the ODT with five odors assigned as target odors, and one odor assigned as a non-target odor. During each trial, an odor was quasi-randomly selected for presentation. Each odor appeared once every six trials, such that there was a 5:1 trial ratio of target and non-target trials. Subjects were required to enter and remain in the sampling port for 0.50 s to initiate a trial. The odor valve for the programmed odorant then relaxed, allowing airflow through the odorant. Once the subject remained in the sampling port for a full second, the final valve relaxed allowing odorized air to enter the port. After a 0.5 s odor sampling period, responses to the lick sensor over the next 2 s determined trial outcome. To evaluate responding, the response period was divided into ten 200 ms bins. A response was scored if a lick was detected at the end of one or more of the ten 200 ms response bins. Responses to target odors (“hits”) yielded sucrose delivery while non-responses (“misses”) resulted in a 5 s timeout, during which the house light was extinguished, and a new trial could not be initiated. Similarly, responses during the presentation of the non-target odor (“false alarms”) resulted in a 5 s timeout. There was no consequence for non-responses to the non-target odor (“correct rejections”). Each trial was followed by an inter-trial interval (ITI) of 2 s, which was initiated after reward delivery or timeout from the preceding trial. All ODT training and testing sessions had a duration of 30 min. ODT training was continued until stability was established.

1.4.3. Measures

Measures of interest from the ODT program included: trials completed, short samples, hit rate, correct rejection rate, sensitivity index (1), responsivity index (2), and lick density (pre-response and response). A short sample is scored any time an animal enters the sampling port but, leaves the port before the odor is released for sampling. For each session, lick density was computed separately by trial outcome (e.g., hit, correct rejection). Response lick density is the average percentage of lick bins (10 ms lick bins across the 2 s response period) during which contact with the lick sensor was detected. Pre-response lick density is the same metric computed for the 500 ms sampling period preceding the response period. Subject responses can be monitored throughout trials and graphed on a plot representing the average lick density for all subjects throughout a trial.

$$[\mathrm{SI}=(\mathrm{hit}\mathrm{rate}-\mathrm{false}\mathrm{alarm}\mathrm{rate})/2(\mathrm{hit}\mathrm{rate}+\mathrm{false}\mathrm{alarm}\mathrm{rate})-{(\mathrm{hit}\mathrm{rate}+\mathrm{false}\mathrm{alarm}\mathrm{rate})}^2]$$ (1)

$$[\mathrm{RI}=(\mathrm{hit}\mathrm{rate}+\mathrm{false}\mathrm{alarm}\mathrm{rate})-1/1-{(\mathrm{hit}\mathrm{rate}–\mathrm{false}\mathrm{alarm}\mathrm{rate})}^2]$$ (2)

1.5. Tissue collection and processing

Mice were anesthetized 29 dpi with 87.5 mg/kg Ketamine/12.5 mg/kg Xylazine and transcardially perfused with 0.9% NaCl before the brain was extracted and drop fixed in 4% paraformaldehyde in phosphate buffer overnight. The brain was then transferred to 30% sucrose in phosphate buffered solution (PBS). Coronal sections (40 μm) were sectioned using a freezing sliding microtome (Microm HM430), and sequentially placed in 12 tubes filled with cryoprotective medium (30% glycerin, 30% ethylene glycol, 40% 0.5 M sodium phosphate buffer) and stored at −20 °C until processing. For cresyl violet staining, tissue was mounted and rehydrated before being stained in cresyl violet acetate for 5 min and dehydrated for coverslipping, following standard procedures [6]. This is standard in our laboratory and has been utilized many times to assess tissue damage [27,29].

1.5.1. Quantification procedures

To measure cortical thickness, 10 coronal sections spaced exactly 240 μm apart and stained with cresyl violet were used. These sections were then imaged using a Revolve Pro^® microscope (Echo Laboratories, San Diego, CA) and the infarct and hemisphere volumes were traced using ImageJ software (version 1.51c, 2017). The percent contralateral cortex was then determined (3).

$$[\mathrm{Percent}\mathrm{Contralateral}\mathrm{Cortex}=(\mathrm{Ipsilateral}\mathrm{Cortex}\mathrm{Area}–\mathrm{Infarct}\mathrm{Area})/\mathrm{Contralateral}\mathrm{Cortex}\mathrm{Area}]$$ (3)

1.6. Statistical analyses

Sigmaplot (version 14.5), SPSS (version 27 and 29), and RStudio (version 2021.09.01) were used to perform graphing and statistical analysis. Significance was determined with a p <.05 and values are expressed as the mean ± standard error. For the behavior tasks, a linear mixed effects model was analyzed using RStudio and the lme4 R package [4], where mice were specified as a random factor to help account for unequal group sizes due to mortality. A one-way analysis of variance (ANOVA) was performed to test the differences between surgery groups. Where differences were found, Tukey’s post-hoc test was performed to examine differences between the surgery groups.

Olfactometry results were computed using SPSS. To determine if stroke affected performance on the ODT across time, Surgery and Sex, a three-way (2 ×3×2) mixed ANOVA was used. Task performance was compared between baseline (day before stroke) and 1 DPI. The main effects of Time, Surgery, and Sex were investigated for each response parameter: trials completed, short samples, hit rate, correct rejection rate, SI, RI, pre-response and response lick density of hit trials, as well as pre-response and response lick density of correct rejection trials. Interactions involving two between-subjects factors were decomposed by pairwise comparisons. Interactions involving a between and within-subjects factor were decomposed by completing one-way ANOVAs within groups and/or paired samples t-tests.

Mixed modeling was used to evaluate changes in performance over the post-stroke recovery period (sessions 1–20). A multilevel modeling approach was implemented to account for nested data from each subject providing multiple measures recorded from the ODT across sessions post-surgery. Measures from the ODT were modeled with session, sex, and surgery group as covariates. Session was modeled as a level 1 predictor, whereas surgery group was modeled as a level 2 predictor. Cross-level interactions were modeled for the session and surgery group. The missing data rate was 6% across all outcome variables. Outliers were defined as three standard deviations from the mean and were winsorized to this value. Arc sine transformations were applied to HR, CRR, and RI, and a log transformation was applied to SI.

2. Results

2.1. Weight

Baseline weight was taken prior to surgery, and used to determine percent change at 1-, 3-, 7-, 14-, and 28 DPI (Table 1). There was a significant main effect of Time (F _{(2.19, 94.2)} = 14.2, p <.0001) and Surgery (F _{(2, 43)} = 5.70, p <.0001, p = .0064). There was also a significant interaction between Time and Surgery on weight change (F _{(8, 172)} = 2.43, p <.0001). Post-hoc analysis revealed that the PTS group lost significantly more weight at 1- (p = .024), 3- (p = .006), and 7 DPI (p = .035) compared to Sham animals. The PTS group also exhibited increased weight loss 1- (p = .035), 3- (p = .031), and 7 DPI (p = .041) compared to the dMCAO group. Additionally, the dMCAO group exhibited increased weight loss at 3 DPI (p = .022) compared to the Sham animals. There were no other significant differences observed at later time points (p >.05).

Table 1.

Summary of both Male and Female weight data and motor functional recovery assessments.

	Male							Female
		1	3	7	14	28	F(df), p-value		1	3	7	14	28	F(df), p-value
Weight Change	Sham	3.25% (± 2.58)	5.13% (3.12)	6.75% (± 3.28)	4.25% (± 2.92)	6.00% (± 3.15)	F (8, 80) = 1.11, p =.365	Sham	1.38% (± 1.16)	3.50% (± 1.10)	3.38% (± 1.55)	8.13% (± 1.99)	2.13% (1.32)	F (8, 80) = 1.54, p =.157
	PTS	−9.50% (± 6.43)	−10.17% (± 7.26)	−5.83% (± 6.42)	−5.00% (± 7.70)	−2.33% (± 5.68)		PTS	−5.57% (± 1.63)	−8.00% (± 1.73)	−3.14% (± 181)	4.29% (± 1.89)	0.14% (± 1.99)
	dMCAO	0.56% (± 1.37)	1.33% (± 1.35)	4.56% (2.26)	2.11% (± 1.82)	4.22% (± 2.49)		dMCAO	−0.75% (± 119)	−2.75% (± 1.92)	1.13% (± 1.61)	5.63% (± 2.32)	2.38% (± 1.69)
Cylinder Task	Sham	−0.12 (± 0.1)	−0.16 (± 0.06)	−0.05 (± 0.04)	0.11 (± 0.14)	−0.1 (± 0.07)	F (8, 73) = 4.85, p <.0001	Sham	−0.28 (± 0.12)	−0.25 (± 0.14)	−0.17 (± 0.12)	−0.09 (± 0.08)	−0.03 (± 0.09)	F (8, 72) = 1.34, p =.237
	PTS	0.24 (± 0.26)	−0.73 (± 0.27	−0.11 (± 0.11)	−0.09 (± 0.11)	0.07 (± 0.08)		PTS	−0.11 (± 0.25)	−0.43 (± 0.21)	0.02 (± 0.11)	0.06 (± 0.07)	0.09 (± 0.05)
	dMCAO	−0.03 (± 0.07)	−0.02 (± 0.07)	−0.01 (± 0.03)	−0.03 (± 0.04)	−0.06 (± 0.07)		dMCAO	−0.17 (± 0.14)	0.04 (± 0.09)	0.10 (± 0.06)	−0.05 (± 0.06)	−0.04 (± 0.04)
Grid Walk	Sham	3.00 (± 1.60)	2.25 (± 0.56)	1.75 (± 0.67)	1.63 (± 0.42)	1.00 (± 0.38)	F (8, 80) = 1.39, p =.216	Sham	3.38 (± 0.80)	1.88 (± 0.69)	2.13 (± 0.44)	1.38 (± 0.38)	1.25 (± 0.37)	F (8, 79) = 6.73, p <.0001
	PTS	8.53 (± 2.61)	5.50 (± 0.99)	5.50 (± 1.15)	3.13 (0.79)	2.33 (± 0.33)		PTS	18.79 (± 4.72)	8.26 (± 1.37)	4.57 (± 0.97)	5.86 (± 0.80)	3.71 (± 0.78)
	dMCAO	3.97 (± 1.21)	2.11 (± 0.39)	2.56 (± 0.63)	2.11 (± 0.26)	2.33 (± 0.44)		dMCAO	4.13 (± 1.08)	2.50 (± 1.30)	1.63 (± 0.50)	2.00 (± 0.53)	1.88 (± 0.58)
Tapered Beam	Sham	0.44 (± 0.15)	0.26 (± 0.08)	0.69 (± 0.26)	0.51 (± 0.15)	0.61 (± 0.14	F (8, 76) = 5.65, p <.0001	Sham	0.16 (± 0.05)	0.10 (± 0.05)	0.15 (± 0.08)	0.23 (± 0.13)	0.16 (± 0.09)	F (8, 80) = 10.9, p <.0001
	PTS	3.83 (± 1.21	3.48 (± 1.21)	1.80 (± 0.44)	1.37 (± 0.44)	1.07 (± 0.38)		PTS	3.71 (± 0.88)	4.29 (± 0.89)	2.41 (± 0.83)	0.51 (± 0.18)	0.79 (± 0.34)
	dMCAO	0.40 (± 0.11)	0.19 (± 0.06)	0.10 (± 0.05)	0.48 (± 0.23)	0.27 (± 0.12)		dMCAO	0.18 (± 0.05)	0.13 (± 0.05)	0.21 (± 0.07)	0.30 (± 0.11)	0.14 (± 0.09)
Rotating Beam	Sham	104.25 (± 5.35)	111.09 (± 3.91)	114.09 (± 0.91)	106.79 (± 5.39	102.88 (± 8.58	F (8, 80) = 7.94, p <.0001	Sham	114.59 (± 0.41)	112.50 (± 2.50)	115 (± 0.00)	112.16 (± 2.11)	110.84 (± 4.16)	F (8, 80) = 12.7, p <.0001
	PTS	8.88 (± 2.21)	55.17 (± 22.10)	31.78 (± 9.59)	95.72 (± 6.64)	97.72 (± 11.17)		PTS	15.69 (± 6.41)	9.34 (± 2.58)	52.90 (± 18.25)	94.10 (± 11.27)	97.96 (± 9.17)
	dMCAO	98.67 (± 7.19)	99.11 (± 9.59)	112.59 (± 2.41)	106.59 (± 5.08)	100.74 (± 6.64)		dMCAO	103.71 (± 5.54)	112.68 (± 1.90)	112.38 (± 2.63)	101.63 (± 12.91)	104.30 (± 4.59)

Note: Comparison of Surgery separated by Sex on percent weight change and motor function assessments. F statistics are represented by interaction between Surgery and Time within each sex. Values are represented values as mean standard error.* p < .05, ** p < .01, *** p < .001.

We evaluated behavioral recovery using four motor tasks (cylinder, grid walk, tapered beam, and rotating beam) at multiple time points (1-, 3-, 7-, 14-, and 28 dpi). Weight Change: The female PTS surgery group exhibited weight loss at 1 dpi (p < .05), 3 dpi (p < .001), and 7 dpi (p < .05) compared to the female sham surgery group. The dMCAO surgery group also experienced weight loss at 7 dpi (p < .05) compared to the female sham surgery. Regardless of sex, all surgery groups showed weight gain in the days following stroke induction, starting around 7 dpi. Cylinder Task: There were no differences observed in the cylinder task. Grid Walk: The PTS surgery group had an increased number of foot faults compared to Sham at 3 dpi (p < .05). The female PTS surgery group exhibited more foot faults than the male PTS surgery group at 14 dpi (p < .05). The female PTS surgery group showed improved functional recovery, with fewer foot faults starting at 3 dpi (p < .05). Tapered Beam: The PTS surgery group had more errors than dMCAO and Sham. For females in the PTS surgery group, there were more errors compared to Sham at 1 dpi (p < .01), 3 dpi (p < .01), and 7 dpi (p < .05). Females in the PTS surgery group also exhibited more errors compared to the dMCAO surgery group at 1 dpi (p < .01), 3 dpi (p < .01), and 7 dpi (p < .05). No differences were detected between sham and dMCAO, regardless of sex. Rotating Beam: Both males and females in the PTS surgery traveled less distance than sham and dMCAO mice at 1- (males, p <.001; females, p < .001), 3- (females, p < .001), and 7 (males, p < .001; females, p < .05) dpi. There were no differences between sex within surgery groups. However, there was an improvement in functional recovery in the PTS surgery group by 14 dpi. The sham and dMCAO surgery groups showed no differences in distance traveled regardless of sex. Statistics are reported as F values that represent the interaction between Surgery and Time for each Sex. Surgery differences are compared to sham (*p <.05, **p <.01, ***p < .001) or dMCAO (#p < .05, ##p < .01, ###p < .001). Sex differences are compared to female mice ($ p < .05) with Surgery group. Time differences are compared to 1 dpi († p < .05, ††p < .01, †††p < .001) within Surgery group. Values are represented as Mean ± SEM. Acronyms: PTS, photothrombotic; dMCAO, distal middle cerebral artery occlusion; dpi, days post injury.

2.2. Cylinder Task

A significant main effect of Time (F _{(4.40, 169)} = 5.92, p = .0001) was found on the cylinder task, but there was no significant main effect of Surgery (F _{(2, 43)} = 0.54, p = .589, Fig. 1B). The interaction between Time and Surgery was significant (F _{(14, 269)} = 3.27, p < .0001). Post-hoc analyses revealed that the PTS performed significantly worse than the dMCAO group at 3 DPI (p = .029). There were no other significant main effects or interactions among the groups at any other time point.

2.3. Grid Walk

For percentage of footfalls on the grid walk (Fig. 1C), results of the mixed-effects model analysis identified significant main effects of Time (F _{(1.94, 83.3)} = 24.9, p < .0001) and Surgery (F _{(2, 43)} = 22.5, p < .0001). There was also a significant interaction between Time and Surgery (F _{(14, 300)} = 8.20, p < .0001). Post-hoc analyses revealed the PTS group performed significantly worse than the Sham group 1- (p = .014), 3- (p = .0004), 7- (p = .004), 14- (p = .0021), and 28 DPI (p = .005, Fig. 1C). There were also significant differences between the PTS and dMCAO group 1- (p = .023), 3- (p = .001), 7- (p = .007), and 14 DPI (p = .008), but not 28 DPI (p = .255). There were no other significant main effects or interactions among the groups at any other time point.

2.4. Tapered beam

The mixed-effects model for the average number of total contralateral limb errors on tapered beam returned significant main effects of Time (F _{(3.47, 147)} = 21.1, p <.0001) and Surgery (F _{(2, 43)} = 44.7, p < .0001, Fig. 1D). The interaction effect between Time and Surgery was also significant (F _{(14, 296)} = 20.9, p < .0001). Post-hoc analyses revealed significantly more errors produced by the PTS group as compared with Sham on 1- (p = .001), 3- (p = .0005), and 7 DPI (p = .011). Additionally, there were significantly more errors observed for the PTS than the dMCAO group on 1- (p = .001), 3- (p = .0005), 7- (p = .004), and 28 DPI (p = .04). There were no other significant main effects or interactions among the groups at any other time point.

2.5. Rotating beam

There were significant effects of both Time (F _{(3.83, 165)} = 37.0, p < .0001) and Surgery (F _{(2, 43)} = 111, p < .0001) regarding the average distance to fall off the rotating beam (Fig. 1E). There was also a significant interaction between Time and Surgery (F _{(14, 301)} = 27.0, p < .0001). Post-hoc analyses demonstrated that PTS fell at shorter distances as compared with both Sham and dMCAO at 1-, 3-, and 7 DPI, (p < .0001). There were no other significant main effects or interactions among the groups at any other time point.

2.6. 5:1 ODT

On average, subjects completed “head-entry” training after an average of 2.4 sessions (range = 1–11) and “head-hold” training after an average of 2.83 sessions (range = 1–4). Subjects were then trained on the ODT task for an average of 30 sessions. Mean SI for each group over the six days preceding surgery were as follows: Sham = 77 (SD = .09), dMCAO = 76 (SD = .06), and PTS = 70 (SD = .03). A majority of subjects (86.67%) met a stability criterion of having a less than a 15% change between the first and latter half of the final six sessions before surgery. Five subjects were excluded from the pre-post analysis of SI, RI, HR, CRR and lick density measures as they completed fewer than six trials on 1 DPI. Each of these subjects were in the PTS group. Three additional subjects were excluded from the analysis of CR lick density due to completing less than three CR trials (two PTS, one dMCAO).

2.7. Sensitivity Index (SI)

Pre-post analysis of SI yielded a significant main effect of Time (F _{(1, 34)} = 17.56, p = .001), with SI being lower for all subject’s post-surgery (Fig. 2A). A main effect of Surgery was also observed (F _{(2, 34)} = 3.73, p = .034) with PTS having significantly lower SI than Sham (p = .01). No other significant main effects or interactions of Time, Surgery, or Sex were observed (all ps >.05). SI significantly improved over the recovery period (t (71.30) = 5.36, p = . 009, Fig. 3A). There was no significant main effect of Surgery, nor an interaction effect of Surgery moderating the association between session and SI during the recovery period (ps > .05). The multilevel model explained significantly more variance than the null model (p < .001, pseudo r² = .04).

Fig. 2.

Photothrombotic stroke worsened performance on the ODT in the first session post-surgery. (A) All subjects exhibited significantly reduced sensitivity index post-surgery (F = 17.556, p < .001). Additionally, the PTS mice exhibited significantly lower sensitivity index compared to Sham (F = 3.731, p = .034). There was no difference between dMCAO and Sham. (B) All subjects exhibited significantly more liberal responding post-surgery (F = 9.264, p = .004). Additionally, the PTS mice exhibited significantly more liberal responding when compared to Sham and dMCAO (F = 3.337, p = .048). There was no difference between dMCAO and Sham. Additionally, a significant Time × Sex × Surgery interaction was observed (F _{(2, 34)} = 4.621, p = .017) and is depicted in Fig. 4 (C) All subjects exhibited significantly reduced trials completed post-surgery (F = 77.906, p < .001). PTS mice completed significantly less trials post-surgery when compared to Sham and dMCAO (F = 5.953, p = .006). There was no difference between dMCAO and Sham. (D) All subjects exhibited significantly reduced short samples following surgery (F = 6.259, p = .017). There were no other significant main effects or interactions. (E) All subjects exhibited significantly reduced hit rate post-surgery (F = 8.686, p = .006). There were no other significant main effects or interactions. (F) All subjects exhibited significantly reduced correct rejection rate post-surgery (F = 17.556, p < .001). Additionally, the PTS mice exhibited significantly lower correct rejection rate when compared to Sham (F = 3.354 p = .047). Additionally, a significant Time × Surgery × Sex interaction was observed (F _{(2, 34)} = 4.355 p = .021) and is depicted in Fig. 4. Error bars represent mean standard error. Abbreviations: PTS, Photothrombotic; dMCAO, distal middle cerebral artery occlusion.

Fig. 3.

All subjects improved on the ODT following surgery. (A) For all subjects, sensitivity index significantly improved over Time (t (71.30) = 5.36, p < .01). There were no other significant main effects or interactions. (B) For all subjects, responding becoming more conservative over Time (t (707.40) = 3.03, p < .01). There were no other significant main effects or interactions. (C) For all subjects, trials completed significantly increased across sessions (t (737.96) = 5.40, p < .01). There were no other significant main effects or interactions. (D) For all subjects, short samples significantly decreased over time (t (735.95) = −3.51, p < .01). Additionally, PTS subjects exhibited significantly fewer short samples than the Sham group (t (78.47) = −2.04, p = .04). Short samples for dMCAO did not differ from PTS or Sham. Across Time, short samples significantly decreased for Sham (t (735.45) = 2.58, p = .01) while no change was observed for PTS condition and dMCAO. (E) There were no significant main effects or interactions for hit rate over Time (ps > .05). (F) For all subjects, correct rejection rate significantly decreased over Time (t (734.57) = −2.72, p < .01) PTS mice exhibited lower correct rejection rate compared with dMCAO (t (206.11) = 3.38, p < .01) and Sham (t (206.11) = 2.29, p = .02). Sham mice exhibited lower correct rejection rate over time (t (734.57) = 1.99, p = .04) compared to the PTS group for whom correct rejection rate did not change across sessions. Change in correct rejection rate across sessions for dMCAO mice did not differ significantly from Sham or PTS. Error bars represent mean standard error. Abbreviations: PTS, Photothrombotic, dMCAO, distal middle cerebral artery occlusion.

2.7.1. Responsivity index (RI)

Pre-post analysis of RI (Fig. 2B) yielded a significant main effect of Time (F _{(1, 34)} = 9.26, p = .004) with more liberal responding observed post-surgery. A significant main effect of Surgery was also observed (F _{(2, 34)} = 3.34, p = .048) with more liberal responding produced by PTS compared with dMCAO (p = .030) and Sham (p = .021). Additionally, a significant Time × Sex × Surgery interaction was observed (F _{(2, 34)} = 4.62, p = .017, Fig. 4). A Time × Surgery ANOVA on the performance of Females yielded no significant main effects or interactions (ps > .05; Fig. 4A). Amongst Males, there was a significant main effect of Time, (F _{(1, 18)} = 14.57, p = .001), as well as a Time × Surgery interaction (F _{(2, 18)} = 7.40, p = .005, Fig. 4B). Simple effects analysis revealed that RI significantly increased post-surgery for both the PTS (p = .007) and dMCAO groups (p = .048), but not Sham (p =. 416). Over the recovery period (Fig. 3B), RI significantly covaried with session (t (707.40) = 3.03, p = .002), with responding becoming more conservative across Time. There was no significant main effect of Surgery group, nor an interaction effect of Surgery moderating the association between Time and RI (ps > .05). The model explained significantly more variance than the null model (p < .001, pseudo r² = .08).

Fig. 4.

Males receiving a stroke responded more liberally and less accurately on the ODT than females. (A) No significant main effects or interactions were found for female responsivity index. (B) Males had significantly more liberal responding post-surgery for PTS and dMCAO groups, but not sham (F = 7.404, p = .005). (C) No significant main effects or interactions were found for female correct rejection rate. (D) Males had significantly lower correct rejection rate post-surgery for PTS and dMCAO groups, but not sham (F = 7.105, p = .005). Error bars represent mean standard error. * p < .05 (PTS compared to Sham). Abbreviations: PTS, Photothrombotic; dMCAO, distal middle cerebral artery occlusion.

2.7.2. Trials completed

The pre-post analysis of completed trials, yielded a significant main effect of Time (F _{(1, 39)} = 77.906, p < .001) with reductions post-surgery for all subjects (Fig. 2C). A main effect of Surgery was also observed, (F _{(2, 39)} = 5.953, p = .006) with PTS completing fewer trials than Sham (p =.003) and dMCAO (p = .007). No other significant main effects or interactions between Time, Surgery, or Sex were observed (all ps > .05). Over the recovery period, trials completed significantly increased across sessions (t (737.96) = 5.40, p < .001, Fig. 3C). There was no significant main effect of Surgery nor an interaction between Surgery and Time (ps > .05). The multilevel model explained significantly more variance than the null model (p < .001, pseudo r² = .60)

2.7.3. Hit rate (HR)

Pre-post analysis of HR yielded a significant main effect of Time, (F _{(1, 34)} = 8.686, p = .006) with lower rates observed post-surgery (Fig. 2E). No other significant main effects or interactions were observed (all ps > .05). Over the recovery period, HR did not significantly covary with session, and there was no significant main effect of Surgery or an interaction effect of Surgery moderating the association between session and HR (ps > .05, Fig. 3E).

2.7.4. Correct rejection rate (CRR)

Pre-post analysis of CRR yielded a significant main effect of Time, (F _{(1, 34)} = 17.556, p < .001) with CRR being lower post-surgery (Fig. 2F). There was a significant main effect of Surgery, (F _{(2, 34)} = 3.354 p = .047) with CRR lower for PTS compared with Sham (p = .010) and dMCAO (p = .033). A significant Time × Surgery × Sex interaction was also observed (F _{(2, 34)} = 4.355 p = .021, Fig. 4). A Time × Surgery ANOVA on the performance of females yielded no significant main effects or interactions (ps > .05, Fig. 4C). Amongst Males, there was a significant main effect of Time (F _(1,18) = 23.240, p < .001), as well as a Time × Surgery interaction (F _{(2, 18)} = 7.105, p = .005, Fig. 4D). Simple effects analysis revealed that CRR significantly decreased post-surgery for both the dMCAO (p = .023) and PTS (p = .014) groups, but not Sham (p = .078). Over the recovery period (Fig. 3F), CRR significantly covaried with session (t (734.57) = −2.72, p = .007), decreasing across sessions. There was a significant main effect of Surgery group with the PTS group producing a lower CRR compared with dMCAO (t (206.11) = 3.38, p = .001) and Sham (t (206.11) = 2.29, p = .02). There was no significant difference in CRR performance between dMCAO and Sham (p > .05). Additionally, there was an interaction effect of the Surgery group moderating the association between session and CRR. Subject’s CRR decreased across sessions for the Sham group (t (734.57) = 1.99, p = .04), compared to the PTS group for whom CRR did not change across sessions. Change in CRR across sessions for the dMCAO group did not differ significantly from the Sham group or the PTS group (p > .05). The model explained significantly more variance than the null model (p < .001, pseudo r² = .06).

2.7.5. Short samples (SS)

Pre-post analysis of SS demonstrated that SS were significantly reduced post-surgery (F _{(1, 39)} = 6.259, p = .017, Fig. 2D). No other significant main effects or interactions were observed (all ps >.05). Over the recovery period, SS significantly covaried with session (t (735.95) = −3.51, p = .001, Fig. 3D), decreasing across Time. There was a significant main effect of Surgery in which PTS subjects produced significantly fewer SS than the Sham group (t (78.47) = −2.04, p = .04). The dMCAO group did not differ from PTS or Sham groups with regard to SS (p > .05). Additionally, there was an interaction effect of Surgery moderating the association between session and SS (t (735.45) = 2.58, p = .01). Upon decomposing this interaction, SS significantly decreased for Sham (t (78.47) = 2.037, p = .04) while no change was observed for PTS or dMCAO (p > .05). The model explained significantly more variance than the null model (p < .001, pseudo r² = .69).

2.7.6. Lick density of hit trials

For each trial, licks occurring during the first 500 ms after the odor is released, the pre-response period, are not counted towards the response criterion. Pre-post analysis of lick density in the pre-response period yielded a significant main effect of Time (F _{(1, 34)} = 4.255, p = .047) with lick density during the pre-response period being significantly higher post-surgery (Fig. 5A, B). No other significant main effects or interactions were observed (all ps > .05). Pre-post analysis of licking during the response period also yielded a significant main effect of Time (F _{(1, 34)} = 36.930, p < .001), with lick density during the response period being lower after surgery. The Time × Surgery and Time × Surgery × Sex interactions for pre-response lick density approached but did not meet statistical significance (ps of .054 and .096, respectively). No other significant main effects or interactions were observed (all ps > .05). During the recovery period, no main effects or interactions of Time or Surgery were observed for pre-response lick density. With regard to lick density during the recovery period, there was a significant main effect of surgery with lower lick density observed in dMCAO as compared with PTS (t (41.23) = −2.49, p = .02) and Sham (t (41.23) = −2.67, p = .01). Response period lick density did not significantly differ between PTS and Sham (p > .05). There was a significant interaction of surgery moderating the association between lick density and session during the response period (t (712) = 3.17, p = .002). Lick density increased across sessions for dMCAO (p = .011) but showed no significant change in the PTS or Sham groups (p > .05).

Fig. 5.

Licking patterns during the ODT illustrate disrupted fine motor movement following stroke. Subject response can be monitored throughout trials and graphed on a plot representing the average lick density for all subjects throughout a trial. Graphs represent the average licking behavior for Baseline (A & B) and the first session following surgery (C & D). Graphing of lick density forms a curve, which can be viewed to identify the licking pattern across response parameters. On the X-axis of a lick-curve, the 2.5 s of the entire trial is represented by 125 time-blocks (10 ms each). On the Y-axis is the average lick density for that time-block. At the beginning of a trial odor presentation begins. At this point, the subject tends to lick infrequently while waiting on odor delivery. When an odor is delivered, lick density declines as the subject is sniffing. Licking then increases, shortly before the response monitoring period begins. (A) Average lick density across time for hit trials during the baseline session. (B) Average lick density across time for hit trials at 1 d post-injury. C) Average lick density across time for correct rejection trials during the baseline session. (D) Average lick density across time for correct rejection trials at one day post-injury. An increase in licking rate is the response expected for target odors and demonstrates a correct response (a hit). Following surgery, all subjects exhibited significantly higher lick density during the pre-response period (F = 4.255, p = .047) and significantly lower lick density during the response period (F = 36.930, p < .001) of hit trials. Additionally, dMCAO exhibited significantly higher lick density during the response period as compared with PTS (t (41.23) = −2.49, p = .02) and Sham (t (41.23) = −2.67, p = .01) for hit trials. Lick density for hit trials increased across sessions for dMCAO but showed no significant change in the PTS or Sham groups (p > .05). (E) Following surgery, all subjects exhibited lower response lick density during correct rejection trials (F = 6.142, p = .019). Abbreviations: PTS, Photothrombotic; dMCAO, distal middle cerebral artery occlusion; CR, correct rejections.

2.7.7. Lick density of correct rejection trials

No significant main effects or interactions were observed for pre-response lick density during correct rejection trials (all ps > .05, Fig. 5C, D). During the response period, a significant main effect of Time (F _{(1, 31)} = 6.142, p = .019) was confirmed with significantly lower lick density post-surgery. No other significant main effects or interactions were observed (all ps > .05). During the recovery period, there were no significant main effects or interactions between Surgery and session with regard to pre-response lick density (ps > .05). With regard to lick density during the response period, no significant main effects or interactions were observed (ps > .05).

2.7.8. Validation of infarct size and location across dMCAO and PTS models

To validate that the dMCAO and PTS models of ischemia, we used Cresyl Violet as a nissl stain to identify and quantify the infarct volume. A one-way ANOVA was computed separately for males and females. For males, there was a significant main effect of surgery group (F _{(2, 22)} = 40.25, p < .001). Twenty-nine days post-stroke, there were no differences in the infarct size between the PTS and dMCAO stroke models (p = .48). However, both the PTS (p < .001) and the dMCAO (p < .001) groups had a significant reduction of cortical tissue in the ipsilateral area of the stroke compared to the Sham group. For females, there was also a significant main effect of surgery group (F _{(2, 22)} = 26.28, p < .001). Twenty-nine days post-stroke, there were no differences in the infarct size between the PTS and dMCAO stroke models (p = 1.00). However, both the PTS (p < .001) and the dMCAO (p < .001) groups had a significant reduction of cortical tissue in the ipsilateral area of the stroke compared to the Sham group.

3. Discussion

The current study sought to evaluate the time course of effects from two commonly used stroke models, dMCAO and PTS, on automated and manually scored tasks of motor behavior and cognition. As expected, PTS induction targeted at the motor strip yielded deficits in motor control, relative to Sham, which were not observed with dMCAO. Observed deficits for PTS were particularly profound for average distance until falling on the rotating beam, percentage of foot faults on the grid walk, and average errors for contralateral limbs on the tapered beam. Disruption of grid walk performance was the most enduring, with differences between PTS and Sham observable at the latest assessment point (28 DPI). In contrast, disruption of tapered beam and rotating beam were detectable for no longer than one week. While these tasks were clearly sensitive to disruption of motor behavior produced by stroke induction, a limitation of these tasks is that they are considerably time-intensive to conduct and score. Thus, an automated task providing measures of both motor behavior and cognition, the ODT, was assessed to determine if its measures are similarly sensitive to the effects of PTS and dMCAO.

With regard to ODT performance, both stroke induction and sham surgery led to significant reductions in trials completed. While the moderating effect of surgery group on reductions in trial count did not reach significance, the PTS group appeared to be particularly impacted with five PTS subjects failing to complete enough trials at 1 DPI for signal detection measures (SI and RI) to be computed. For all surgery groups (including Sham), disruption of trial count was relatively long-lived, approaching baseline levels only in the final week of testing. However, trial count did not distinguish PTS from dMCAO or Sham beyond the exceptionally low trial counts observed in numerous PTS subjects at 1 DPI. Surgery also resulted in reductions in HR and SI for all groups at 1 DPI. Again, while a larger deficit of SI was observed with PTS, the moderating effect of surgery group did not reach significance.

Interestingly, the effects of surgery on RI and CRR were both moderated by an interaction of sex and surgery group. Only males in the PTS group demonstrated significant changes at 1 DPI, with responding becoming more liberal and yielding a lower CRR. Sex differences have been noted in the neurophysiological response to PTS [19,25,30]. The present study observed a sex-dependent behavioral response as well, underscoring the need to assess for sex-dependent effects in preclinical studies of stroke, as noted by other authors [25].

It appears likely that the impact of surgery on RI and CRR in males of the PTS group was driven by disruption of motor control for licking. As mentioned, the pattern of lick responding for the PTS group post-surgery deviated from the sigmoidal shape typically observed performance of the task. The interactions for pre-response lick density trended towards but, did not reach statistical significance. Yet, this appears to have manifested in difficulty inhibiting licking on non-target trials, yielding a significant deficit in CRR amongst PTS subjects.

The ability to evaluate motor components of responding by assessing lick patterns is a unique feature of the ODT. Lick density measures were clearly not as sensitive to the disruption of motor behavior produced by PTS as alternative tasks such as the grid walk, cylinder task, tapered beam, and rotating beam. Despite the lack of sensitivity of the ODT lick measures for the gross disruption of motor behavior produced by PTS, these measures may have particular utility for evaluating dysphagia produced by stroke. Analysis of licking behavior is a common means by which dysphagia is evaluated in mouse models [18]. Typically, this is performed by evaluating the licking behavior produced when consuming water. In the ODT, the timing of licking is explicitly trained. As such, more subtle deviations from an intended licking pattern may be detected.

Deficits in oromotor control may have also contributed to the lower weights observed in PTS subjects following surgery compared with Sham and dMCAO. It is important to note that all animals were maintained on a food deprivation protocol intended to maintain them at 85–90% of their free-feeding weight. The feed amount delivered each day was determined by an evaluation of the weight of each animal in the box. However, the food was ultimately administered to the full box of animals, which included subjects from each of the experimental groups. As a result of the pronounced motor deficits, PTS subjects were likely less efficient in consuming their food, and/or defending their ration from cage mates, as compared with Sham or dMCAO subjects. Group differences in weight normalized within two-weeks, consistent with the time course of PTS deficits on the tapered beam and rotating beam tasks.

The most profound effect surgery produced on the ODT was a deficit in task motivation, as evidenced by decreased trial count. Though the effect was most pronounced in the PTS condition, this was observed in the Sham animals as well. This demonstrates that aspects of the Sham procedure, such as the administration of Rose Bengal, Buprenorphine, Cephazolin, or general anesthesia, produces acute behavioral effects detectable through ODT measures. This is a limitation that needs to be considered in investigations of stroke.

The ODT task was used to assess attention, impulsivity, motivation, and motor impairment. The use of the ODT offers the ability to measure motor and cognitive behavior with high temporal precision and behavioral sensitivity. Using traditional behavioral tasks (i.e., cylinder task, grid walk, tapered beam, and rotating beam) and comparing performance with the ODT allowed validation of the ODT as a task sensitive to acute motor impairment. Through the use of multiple tasks, the current study captured effects of impaired behavior of subjects who received a PTS in the form of both gross motor ability and fine motor behavior. The current study also analyzed long-term effects of stroke by utilizing a multilevel modeling approach of performance in the recovery period (sessions 1–20).

Over the recovery period, trials completed and SI increased across all surgery groups. Trials completed and SI were lowest after surgery and these improved as time after surgery increased. RI became more conservative over the recovery period across surgery groups, demonstrating reductions of bias in responding. HR remained stable over the recovery period. Given the acute post-surgery deficits observed, these findings were consistent with functional recovery as expected. CRR and SS decreased across the recovery period for sham, but showed no significant change over the recovery period for dMCAO and PTS. Lick density of hit trials during the response period increased across the recovery period for dMCAO, while showing no change for sham and PTS. Based on the minor improvement in the model variance, these significant but unexpected effects were quite subtle.

Both PTS and dMCAO stroke models are commonly used in stroke research, with benefits and disadvantages for each. The PTS model is known to produce small lesion sizes, but the ability to control where the lesion occurs is an advantage when it is necessary to inflict injury in a particular area, such as the motor cortex [20]. The dMCAO model provides slightly less consistent lesion sizes and infarct borders, though lesion size is dependent on mouse strain. For example, this model produces smaller lesion sizes in C57BL/6J than the BALB/cJ strain [10,20,27]. The natural variability of the MCA can make targeting a particular area of the brain difficult. We found tissue damage of similar size and location for each stroke models as reported previously [10,20,27].

Motor coordination is typically more impaired using the motor cortex PTS model when compared with dMCAO stroke model [11,27]. Our behavior results following a permanent ischemic stroke is in line with the literature, where the PTS model often performed worse than dMCAO and Sham on all of the tasks, while dMCAO and Sham group had no notable differences in task performance. This helps illustrate the necessary use of the PTS model when examining behavioral deficits after stroke.

Evidence of the PTS model as more damaging to motor ability is also observed by examination of ODT performance. Animals in the PTS condition completed significantly less trials, had significantly lower SI, significantly higher RI, and significantly lower CR rate than subjects receiving Sham and dMCAO manipulations. In examining lick density, PTS trended toward higher lick density during the pre-response period and lower lick density during the response period for hit trials, suggesting disrupted motor behavior. Disruption of the lick response in the PTS condition suggests that deficits in accuracy measures are most likely accounted for by motor disruption. Further, 31% of the subjects in the PTS condition were excluded from the analyses of multiple measures comparing baseline and 1 DPI due to completing less than three target and/or non-target trials; 54% of PTS subjects were excluded from the lick density analyses of correct rejection trials analysis for the same reason. The inability of PTS subjects to complete trials demonstrates that motor ability was severely impaired. Thus, impairment on all cognitive measures is attributed to motor impairment, rather than a specific cognitive impairment. The ability to differentiate motor performance and cognitive performance is a major benefit of the ODT.

PTS mice were able to recover observable deficits on most of the tasks in a fairly short time period. On the tapered beam, the number of hindlimb foot faults made was recovered after 7 DPI. This recovery was also examined with the rotating beam, where after 7 DPI, PTS mice were able to travel a similar distance compared to the dMCAO and Sham mice. The grid walk was the most sensitive test examining post-stroke deficits, where differences were detected in the PTS group up to 28 DPI. The rapid recovery of observable deficits in mice is commonly observed, principally in the dMCAO model, and contrasts with the slow and often incomplete recovery typically observed in humans after an ischemic stroke of the middle cerebral artery [15]. The major advantage of the PTS model is the longer duration of observable deficits, but at the cost of clinical relevancy when compared to the dMCAO.

The majority of subjects demonstrated six-day stability of performance on the ODT within 36 sessions of total training (all phases). Many subjects reached six-day stability well before 36 sessions, but continued to receive training until the planned date of surgery. Of rodent tasks of cognition, this is a relatively quick timeline for training and stable performance. In tasks used with animals, particularly rodents, quick training times are imperative for a variety of reasons, especially when considering the lifespan of the typical rodent. It has been demonstrated that rats were able to complete a visual match-to-sample task with 80% accuracy after 25 sessions of training (over 2,500 trials) and reach 90% accuracy after about 50 sessions [16]. Rats completing an olfactory-based match-to-sample task reached 90% accuracy in 24–31 sessions, approximately 576–720 trials [28]. Thus, olfactory stimuli can facilitate stimulus control in task training and testing in a brief manner. Use of olfactory stimuli for the very commonly used task of attention, the 5C-CPT, can speed training time requirements in a more naturally relevant paradigm to rodents. Use of the ODT (an olfactory based adaptation of the 5C-CPT) in the current study illustrates that operant training using olfactory stimuli can be accomplished rapidly and yield high levels of performance.

The ODT is also useful because it allows for assessment of cognition and motor ability concurrently. As shown in the current study, motor impairments can cause disruptions in cognitive performance. The ability to compare cognitive and motor performance in the same task can allow for the differentiation of these effects. The ODT is also automated, allowing for standardized administration, scoring, and interpretation.

3.1. Limitations

We have identified several considerations and limitations to the findings presented here. Due to the use of only one olfactometer, four cohorts of subjects were tested over a period of two years. This leads to the increased possibility of third variables, such as differences in handling, stroke procedure, or olfactory training impacting performance. Additionally, no pure control group was used. The Sham group received anesthesia, Rose Bengal, Buprenorphine, Cephazolin, and an incision, which clearly disrupted performance, as all subjects were significantly impaired on some measures of the ODT during the first post-manipulation session. The use of only one task of cognition, the ODT, does not allow for complete validation of its use for the evaluation of attention. Future research utilizing ODT performance needs to be compared against other validated measures of attention in mice.

Mice in all three groups were under general anesthesia, specifically isoflurane. Isoflurane has been shown to have neuroprotective benefits after stroke induction and may also reduce the true beneficial effects of potential drugs after stroke [33,34]. The consistency in which the PTS model showed worse outcome than both the Sham and dMCAO on behavior tasks is strong evidence that the PTS model is a more useful model for examining behavioral deficits after stroke. The neuroprotective benefits of isoflurane may be masking deficits longitudinally after stroke.

The use of a photoreactive dye to induce stroke in the PTS model allows for precise stroke location, but is less clinically relevant than the dMCAO model in stroke research. The MCA provides blood supply to many different parts of the brain and is the most common artery involved in ischemic stroke [24]. The dMCAO model is therefore a useful and clinically relevant model of stroke targeting the main artery affected by ischemic stroke in humans. Identifying a task that can reliably demonstrate enduring deficits in the dMCAO model can help reduce time, money and the number of animals needed in stroke research.

The present study sought to compare two methods of stroke induction and utilized a range of behavioral tasks to characterize post-surgery effects. We have demonstrated the usefulness of measuring the heterogeneity of deficits found across two commonly used animal models of stroke. The use of two different models of stroke is a strength of the current project. While the results of this effort highlight the strengths and limits of the PTS and dMCAO models, there remains a need for a stroke induction procedure for mice that better reflects the more enduring pattern of post-stroke effects observed in humans. The ongoing use of motor and cognitive assessment in animal models of stroke is needed, given the long-term deficits in both cognition and motor ability are common in humans following stroke. Developing additional tools to assess and evaluate all facets of recovery in animal models of stroke will certainly highlight beneficial effects of therapeutic administration. Ultimately, model improvement will facilitate the development and evaluation of therapeutics intended to prevent or ameliorate stroke-related deficits, improve the standard of medical care and quality of life in humans suffering from a stroke.

Fig. 6.

Representative and quantified verification of infarct analysis. Validation of cortical damage in animals 29 days after induction of dMCAO and PTS, compared to Sham surgery. Representative images of Cresyl Violet-stained coronal brain sections demonstrate damaged cortical tissue 29 days post-stroke in both female (A) and male (B) mice. Quantification of infarct analysis in both dMCAO and PTS models were compared to Sham surgery in both female (C) and male (D) mice. The results showed that both the PTS (p < .001) and dMCAO (p < .001) surgery groups exhibited significant reduction of cortical tissue in the ipsilateral area of the stroke compared to the Sham group. There were no statistical differences in the infarct size between the PTS and dMCAO stroke models (Females, p = 1.00; Males, p = .48). * p < .05, ** p < .01, *** p < .001 (PTS compared to Sham); # p < .05, ## p < .01, ### p < .001 (dMCAO compared to sham). Error bars represent mean standard error. Abbreviations: PTS, Photothrombotic; dMCAO, distal middle cerebral artery occlusion.

Data availability

Data will be made available on request.