Chronic treatment with adenosine A1 receptor antagonist promotes neurogenesis and improves outcome after cerebral ischemia

Abstract

Adenosine A1 receptors (A1ARs) are promising targets for stroke treatment, potentially due to their relatively unexplored effects on proliferation and differentiation of newborn neurons. In this study, we investigated the impact of chronic treatment with the A1ARs antagonist DPCPX on neurogenesis following MCAO in rodents, using PET with [¹⁸F]FLT in rats and immunohistochemistry in mice. In addition, we assessed the therapeutic properties of DPCPX on stroke recovery with a comprehensive battery of neurological and behavioral tests. The outcome shows that blocking A1ARs signaling with DPCPX improved immunohistochemical results in 8 to 28 days after MCAO in mice. PET imaging with [¹⁸F]FLT revealed an increase in cellular proliferation following DPCPX treatment in the subventricular zone at day 8 post-ischemia in rats, a result further supported by IHC in SVZ of ischemic animals. Furthermore, DPCPX enhanced the production and integration of newborn neurons while reducing astrocytic differentiation in the ischemic areas. Finally, behavioral tests showed that chronic treatment with DPCPX ameliorated motor and memory deficits after brain ischemia. All taken in consideration, our results provide novel and compelling evidence of the therapeutic potential of the A1AR antagonist DPCPX for ischemic stroke recovery.

Keywords

A1ARs brain ischemia DPCPX PET imaging neurogenesis

Introduction

Stroke caused by a focal disturbance of the cerebral blood circulation is one of the most devastating diseases in the western world, remaining the leading cause of long-term disabilities. Remarkable progress has been accomplished in acute stroke care through thrombolysis and mechanical thrombectomy in recent years, nevertheless the clinical management of subacute and chronic stroke remains limited due to the lack of effective treatments.^1,2 The disruption of the cerebral blood flow triggers a pathological cascade in the affected neurons, ultimately leading to their death. When neurons die, the release of cytotoxic molecules triggers a continuous increase of adenosine levels, due to the collapse of adenosine triphosphate (ATP) energetic metabolism within minutes to hours after occlusion.^3–5 Although ATP is the most studied energetic metabolite, adenosine also plays a key role in the ischemic cascade.^6,7

Adenosine, an end-product of ATP metabolism, is a key factor in in brain homeostasis, including modulating neurotransmitter release and contributing to synaptic plasticity.^8,9 Likewise, adenosine is an essential nucleoside, and its receptors are widely expressed in the brain, although their distribution exhibits cell type- and region-specific variations.¹⁰ Following cerebral ischemia, extracellular adenosine levels increase, leading to neuronal damage and cell death in the penumbra zone.^4,5,11,12 The activation of the Adenosine A1 receptors (A1ARs), the most expressed adenosine receptors in the brain, has traditionally been described as neuroprotective given their role in providing metabolic support.^13–15 However, the role of these receptors in ischemic stroke is controversial. While some authors describe adenosine activation as beneficial,^6,16,17 others observed detrimental outcomes.^18,19 In addition to this controversy, A1ARs has a major impact on cerebral ischemia, either by activation or inhibition.

In response to ischemic injury, the brain not only exhibits alterations in A1ARs and other signaling pathways but also initiates protective mechanisms to combat neurotoxicity and neuroinflammation. Notably, the subventricular zone (SVZ) plays a well-documented role in mitigating the effects of brain ischemia.^20–23 Neurogenesis occurs primarily in two neurogenic niches in adult mice, the SVZ of the lateral ventricles and the dentate gyrus of the hippocampus.^24,25 The SVZ is the largest of these niches, and sustains the production of newborn neural cells throughout human life. Furthermore, this brain region responds to ischemic injury by generating not only newborn neurons but also new astrocytes.^21,23,26,27 Our laboratory has previously investigated both neurogenesis and gliogenesis following cerebral ischemia using PET imaging with the radiotracer 3′-Deoxy-3′-[¹⁸F]fluorothymidine ([¹⁸F]FLT).^28–30 [¹⁸F]FLT is an analogue of thymidine that, once phosphorylated, cannot be incorporated into DNA and becomes metabolically trapped within cells. For this reason, the uptake and accumulation of [¹⁸F]FLT serve as a surrogate PET imaging marker for cellular proliferation.³¹

Our study aimed to investigate cellular proliferation by [¹⁸F]FLT PET imaging in ischemic animals after being treated with the selective A1ARs antagonist 8-Cyclopentyl-1,3-dipropylxanthine (DPCPX). Chronic administration of DPCPX induced an increase in proliferation at day 8 post-ischemia, mainly in the neurogenic niche of the SVZ. In addition, immunohistochemistry (IHC) was used to evaluate the modulatory effect of A1ARs on proliferation, neuronal differentiation and clinical outcome following ischemic stroke in mice.

The reported results here shed light on the role of A1ARs in brain ischemia and the potential of these receptors as modulators of ischemic stroke outcome. Hence, these findings could pave the way for the development of novel treatments that ultimately improve the clinical management of ischemic stroke.

Materials and methods

Animals

Adult male wild-type C57BL6/J mice (n = 52; 25 ± 2 grams body weight; Janvier, France) and Sprague-Dawley rats (n = 14; 304 ± 7.1 grams body weight; Janvier, France) were used for IHC (mice), behavioral tests (mice) and PET imaging studies (rats). Animal experimental protocols and relevant details regarding welfare were approved by the Comité de Ética en Experimentación Animal (CEEA) at UPV-EHU and CIC biomaGUNE and were conducted in accordance with the Directives of the European Union on animal ethics and welfare (2010/63/UE). Results are reported following the ARRIVE guidelines.³²

Experimental design

Three main experimental scenarios were designed for this study. The first scenario focused on evaluating cellular proliferation using [¹⁸F]FLT PET imaging in rats treated with either DPCPX or a vehicle control (Figure 1(a)). PET imaging sessions were conducted before middle cerebral artery occlusion (MCAO) and at 8, 15 and 28 days after ischemia. A total of 14 rats (n = 7 per treatment group) were included in this experiment. At each time point, five animals per group were scanned, except on day 8, when seven rats per group were scanned. The second scenario, involving 36 mice, focused on investigating biomarkers of proliferation and neurogenesis in ischemic animals treated with vehicle or DPCPX by immunofluorescence (Figure 1(b)). Mice were sacrificed at 8, 15 and 28 days after MCAO. Six animals were used per condition (vehicle or DPCPX) and time point, except for DCX, NeuN and Ki67 quantification where 4 animals were used per condition. Finally, the third experimental scenario was designed to study behavioral changes after vehicle or DPCPX treatment in ischemic mice at 1, 8, 15, 21 and 28 days (Figure 1(c)). To account for potential side effects of DPCPX, sham groups (both vehicle- and DPCPX-treated) were included as controls. Four animals were assigned to each condition, resulting in a total of 16 animals.

Figure 1.

Experimental design. Three experimental scenarios were defined in this study. In the first scenario (a), PET imaging was performed in rats to compare ischemic rats treated with either vehicle or DPCPX. The second scenario (b) involved mice, and immunofluorescence was used to study markers of proliferation and neurogenesis in mice treated with either vehicle or DPCPX (b). The last scenario (c) also used mice to investigate behavioral changes following brain ischemia, with groups treated with either vehicle or DPCPX.

Transient Middle cerebral artery occlusion model (MCAO)

Transient focal ischemia in rats and mice was produced under anesthesia by 90 or 60-min intraluminal occlusion of the middle cerebral artery (MCAO) respectively, followed by reperfusion as described previously.³⁰ In brief, rats and mice were anesthetized with 4% isoflurane in 90% O₂ using an oxygen concentrator (MSS International)_, and maintenance at 1.5–2% isoflurane during surgery. 2.6-cm length of 4–0 monofilament nylon suture for rats and 10 mm length of silicone-coated commercial (602223PK10, Doccol Corporation) for mice were introduced into the right external carotid artery up to the level where the MCA branches out. After occlusion, the filament was removed to allow reperfusion. Following surgery, mice were kept in their cages with free access to food and water. Perioperative and postoperative pain management was conducted using subcutaneous injections of buprenorphine at a dose of 0.05 (rats) or 0.1 (mice) mg/kg. The first dose was administered 15 minutes before MCAO, and subsequent doses were given every 12 hours for the next 72 hours. The animals were subjected to a hydration protocol by subcutaneous injection of saline solution during the first 72 hours and wet diet to improve both rat and mice feeding. Animal wellness was evaluated daily by monitoring body weight and evaluating motor deficit in the animals. Motor deficit was assessed in each animal 1 h after MCAO and later every 24 h. Animals without neurological deficits were excluded from the study. Neurological scores from motor deficits were registered in a system of five-point scale with grade (0: No observable deficit. The animal is active; 1: Failure to extend right paw; 2: Decreased resistance to lateral push and circling to the right; 3: Falling to the right. The animal presents rotating or revolving; 4: Unable to walk spontaneously; 5: Dead animal). The number of animals excluded from this study was 25 in the case of ischemic animals treated with vehicle and 9 in the case of ischemic animals treated with DPCPX due to mortality during different days after reperfusion or due to absence of cerebral infarction.

8-Cyclopentyl-1,3-dipropylxanthine (DPCPX) treatment

Chronic 8-Cyclopentyl-1,3-dipropylxanthine (DPCPX, selective A1ARs antagonist) treatment was carried out every day from MCAO to euthanasia. With that aim, 0.1 mg/kg was injected intraperitoneally once per day over the course of the experiment in a randomized and blinded fashion. In total, four animal groups were studied: sham/MCAO treated with vehicle (PBS 1X) and sham/MCAO treated with DPCPX.

5-bromo-2′-deoxyuridine (BrdU) administration

To label the total pool of proliferative cells after MCAO, we performed three daily injections of 50 mg/kg BrdU, with an interval of 2 hours between injections. BrdU administration was maintained from the MCAO onset until euthanasia.

Radiochemistry

The synthesis of 30-deoxy-30-[¹⁸F]fluorothymidine ([¹⁸F]FLT) was performed as described earlier²⁹ using a TRACERlab FX_FN synthesis module (GE Healthcare). After purification by high performance liquid chromatography (HPLC; stationary phase: VP125/10 Nucleosil 100-7 C18 column, Macherey-Nagel; mobile phase: 0.01 M aqueous NaH2PO4/ethanol, 90/10; retention time: 13–14 min) and sterile filtration, injectable [¹⁸F]FLT solution was obtained with non-decay corrected radiochemical yield of 7.5 ± 1.1% in an overall production time of 62 min. Radiochemical purity was above 95% in all cases at injection.

Positron emission tomography imaging and image analysis

PET scans were performed using an eXplore Vista PET-CT camera (GE Healthcare, Waukesha, WI, USA). Anesthetized animals were placed into a rat holder compatible with the PET acquisition system and maintained in normothermia using a water-based heating blanket at 37°C. To ensure animal welfare, temperature and respiration rate were continuously monitored while they remained in the PET camera, using a SAII M1030 system (SA Instruments, NY, USA). The tail vein was catheterized with a 24-gauge catheter for intravenous administration of the radiotracer. For longitudinal assessment of cellular proliferation, mice were scanned before (day 0) and 8, 15 and 28 days after ischemia with [¹⁸F]FLT. The radiotracer was injected concomitantly with the start of the PET acquisition and dynamic brain images were acquired for 34 frames and 60 min in the 400–700 keV energetic window. After each PET scan, CT acquisitions were also performed (140 mA intensity, 40 kV voltage), to provide anatomical information of each animal as well as the attenuation map for the later PET image reconstruction. Dynamic acquisitions were reconstructed (decay and CT-based attenuation corrected) with filtered back projection (FBP) using a Ramp filter with a cut off frequency of 0.5 mm⁻¹.

PET images were analyzed using PMOD image analysis software (Version 3.5, PMOD Technologies Ltd, Zurich, Switzerland). To verify the anatomical location of the signal, PET images were co-registered to the anatomical data of an MRI rat brain template. Two type of Volumes of Interest (VOIs) were established as follows (Figure 2(b)): (1) A first set of VOIs was defined to study the uptake of [¹⁸F]FLT in SVZ. SVZ VOIs were manually drawn in the both ipsilateral and contralateral hemispheres on slices of the MRI (T2W) rat brain template provided by PMOD software. (2) A second set of VOIs was automatically generated in the cerebral cortex, striatum and hippocampus by using the regions proposed by the PMOD rat brain template, to study the evolution of [¹⁸F]FLT PET signal in these specific regions in ischemic and contralateral hemispheres. The last three frames (last 15 min of acquisition) were averaged to quantify radiotracer uptake. Average values in each ROI were determined and expressed as percentage of injected dose per cubic centimeter (%ID/cc).

Figure 2.

Cellular proliferation was increased in the SVZ at 8 days after the MCAO in DPCPX-treated animals. Experimental set-up of the study (a). Schematics of the VOIs used in PET quantification defined in a MRI rat brain template (b). Representative PET images obtained with [¹⁸F]FLT at 0, 8, 15 and 28 days after MCAO in vehicle (left) and DPCPX-treated animals (right) (c). [¹⁸F]FLT PET signal uptake was quantified as %ID/cc in subventricular zone (d), cortex (e), striatum (f) and hippocampus (g) of the ipsilateral hemisphere. *p < 0.05 and **p < 0.01. Values are presented as scatter dot blot (mean ± SD).

Histology and immunohistochemistry

Mice were anaesthetized with an intraperitoneal injection of pentobarbital (100 mg/kg). Brain tissues were fixed by intracardial perfusion using 4% paraformaldehyde. Fixed brains were sliced at 40 μm of thickness using the Leica VT 1200S vibratome (Leica Microsystems). A cresyl violet staining was performed to assess infarct volume. In brief, tissue slices were incubated in the cresyl violet solution and later dehydrated with an alcohol gradient. Xylol was used as a final step and slices were mounted with Eukit Quick-hardening Mounting Medium (Sigma Aldrich). For immunofluorescence, tissue slices were permeabilized with 0.1% Triton and non-specific epitopes blocked with 1% Bovine Serum Albumin and 10% Donkey serum in PBS. Primary antibodies were incubated overnight and diluted in the same blocking solution. Rabbit α-Doublecortin (Abcam #ab18723), rabbit α-Ki67 (Abcam #ab15580), guinea pig α-NeuN (Synaptic Systems #266004), mouse α-BrdU (Invitrogen #03-3900), rabbit α-A1ARs (alomone #AAR-006), chicken α-GFAP (Abcam #ab4674) and goat α-Thbs4 (R&B systems, #AF2390) were used in this study. After primary antibody incubation, tissue was washed three times with PBS and secondary antibodies were added for one hour. Alexa fluor 594 goat-guinea pig, alexa fluor goat-rabbit 488, alexa fluor goat-mouse 594, alexa fluor 594 donkey-chicken and alexa fluor donkey-goat 488 were used as secondary antibodies. Then, three new washing steps were conducted. Slices were mounted with Mowiol 4-88 Reagent l (Merck). In the BrdU experiments, brain tissue was collected as previously described. Brain slices were incubated for 20 minutes in 2 N HCl to unmask BrdU labeling. After this incubation, the immunofluorescence protocol was carried out as previously described.

Confocal microscopy acquisition and analysis

Confocal microscopy of dual-labeled tissue sections was performed using a Leica TCS SP8 STED CW confocal laser scanning microscope. In the case of MCAO experiments, we selected 6 coronal sections representing the entire striatum. In each section, six regions were analyzed: three from the striatum and three from the cortex, all located near the injured area. Series of optical sections (z-stacks) were imaged (z-step: 8.4 µm) with a 40X (1.0 NA) oil immersion objective and cellular density was calculated as the number of cells/mm². BrdU, Ki67, DCX, NeuN and Thbs4 positive cells were manually counted using the FIJI software (ImageJ).³³ BrdU- and Ki67-positive cells were counted, considering double-positive nuclei for DAPI alongside BrdU or Ki67 markers. For DCX, NeuN and Thbs4, DAPI was used as a control and cells were considered positive when a band surrounded the DAPI staining exceeded a pre-established threshold for each marker.

Behavioral tests

Rotarod test

This test was selected to measure balance and coordination in ischemic mice. Animals were trained twice prior to performing the rotarod test at days 1, 8, 15, 21, and 28 after ischemia. The results represent the average of three evaluations conducted at 10-minute intervals per animal. Latency was defined as the time between the start and the animal's fall. The rotarod protocol was set to progressively increase from 0 to 40 rpm over a 5-minute duration

Pole test

This test assesses basic motor function and has been widely used in animal models of focal ischemia. It effectively differentiates between sham and MCAO animals at both early and relatively late time points. Animals were trained before the ischemic event, and scores were collected every two days after MCAO. The results represent the average of three evaluations conducted at 10-min intervals per animal. Latency was defined as the time between the start and the animal’s fall. Both prolonged latency (when the animal gets stuck) and shortened latency (when the animal falls prematurely) indicate motor deficits.

Corner test

Due to the contralateral nature of the disease, ischemic mice tend to turn towards the non-affected side of the body. This tendency was measured under all conditions at 28 days after MCAO onset to study whether bilateral symmetry was restored following treatment. Mice were placed in a corridor with exits on the right and left. As MCAO model was performed in the left cerebral hemisphere, the tendency to turn to the right was specifically measured.

Cylinder test

This test studies bilateral involvement following MCAO. Animals were placed inside a transparent glass cylinder measuring 40 cm in height and 20 cm in width. The dragging of the front paw of the affected side was observed and recorded. The first twenty attempts were measured. Animals from all conditions were analyzed at 1, 8, 15, 21 and 28 days post-MCAO.

Adhesive removal test

This test evaluates sensorimotor deficits. Animals underwent two training sessions prior to measurements, which were conducted at 1, 8, 15, 21 and 28 days post-MCAO. The time between the first contact of the mouth with the adhesive (sensitivity) and the removal of the adhesive (dexterity) was evaluated.

Novel object recognition

This test examines short-term recognition memory in rodents, leveraging their natural preference for novelty. Animals were trained for 10 minutes using two identical objects immediately before MCAO. Short-term memory was assessed 3 hours post-training and 24 hours later. During evaluation, one object was replaced with a new one, and the time spent with the novel object, as well as the discrimination index were calculated. The discrimination index follows the next equation: ([time exploring the new object-time exploring the familiar object]/total time).

Statistical analysis

Results are presented as means ± SD. Statistical analysis was performed using unpaired Student’s t-test for independent conditions. For comparisons involving more than two groups, one-way, two-way ANOVA or mixed model analysis was performed, followed by Sidak post-hoc test to evaluate differences between groups. The Mann-Whitney U test was performed for Sholl analysis, while the Log-rank (Mantel-Cox) test assessed survival differences among groups. The Chi-square test was used to analyze DCX/Thbs4 proportion and corner test results. The level of significance was set at p < 0.05, and statistical analyses were performed with GraphPad Prism version 8 software.

Results

Effect of chronic treatment with DPCPX on cellular proliferation after brain ischemia

Cellular proliferation was measured by PET imaging at day 0 (control) and at days 8, 15 and 28 after MCAO with the radiotracer [¹⁸F]FLT.^29,30 [¹⁸F]FLT signal uptake (%ID/cc) was studied in the SVZ, ischemic areas (cerebral cortex and striatum) and hippocampus in ischemic rats treated with vehicle (PBS) and DPCPX (Figures 2(a) to (c)). Our results showed a significant increase of [¹⁸F]FLT signal uptake in the SVZ, cortex, and striatum 8 days post-MCAO in rats treated with DPCPX compared to the vehicle group (respectively p < 0.05, p < 0.01 and p < 0.05, Figure 2(d) to (f)) but not in the hippocampus (Figure 2(g)). These results suggested that chronic DPCPX treatment during MCAO increased proliferation in the SVZ and in those brain regions affected by ischemia.

However, no changes were observed in the contralateral hemisphere between vehicle and DPCPX-treated ischemic rats (Figure 1S). Cerebral ischemia induced similar [18F]FLT uptake signals in both hemispheres in SVZ and cerebral cortex, an increase of PET signal in ischemic striatum and a decrease in hippocampus in relation to the contralateral hemisphere (Figures 2 and 1S).

To confirm PET imaging results, we performed BrdU immunofluorescence to label proliferating cells. Chronic BrdU treatment was performed up to 8, 15 and 28 days after brain ischemia onset. We observed an increase of BrdU cells in SVZ and ischemic regions at day 8 after MCAO in DPCPX-treated compared to control ischemic mice (p < 0.01, p < 0.001, Figures 3(a) to (c)). These findings were supported by an increase in Ki67 staining, in both the SVZ and infarcted region 8 days after ischemia in mice treated with DPCPX, compared to control ischemic animals (p < 0.05, p < 0.01, Figures 3(d) to (f)). However, the hippocampus showed no significant differences in Ki67-positive cells between experimental conditions, supporting PET imaging findings following ischemia.

Figure 3.

Immunofluorescence detection of cellular proliferation in SVZ and ischemic area after cerebral ischemia. Representative images of BrdU in MCAO mice treated with vehicle (left) and DPCPX (right) (a). The number of BrdU-positive cells was evaluated at day 8 after ischemia in the SVZ (b) and ischemic area (c). Immunofluorescent images of Ki67-positive cells (pink) and DAPI (black) in ischemic mice treated with vehicle (left) and DPCPX (right) (d). The number of Ki67-positive cells was evaluated at day 8 after ischemia in the SVZ (e), infarcted area (f) and hippocampus (g). *p < 0.05, **p < 0.01 and ***p < 0.001. Scale bars = 500 µm (a) and 50 µm (d). Values are presented as scatter dot blot (mean ± SD).

Differentiation of newborn neurons after DPCPX treatment during the chronic phase of cerebral ischemia

To explore the potential consequences of the increased cellular proliferation in the SVZ after MCAO, induced by chronic DPCPX treatment, we analyzed cellular differentiation in the infarcted areas using immunofluorescence. MCAO groups treated with either vehicle (PBS) or DPCPX were assessed at 0, 8, 15 and 28 days after ischemia with NeuN (mature neurons) and doublecortin (DCX, neuroblast) to label neuronal lineage from the SVZ. In addition, we labelled specifically newborn astrocytes with the thrombospondin 4 (Thbs4) antibody²¹ (Figure 4(a)). Immunofluorescence staining displayed increased differentiation in newborn neurons (in pink, Figure 4(b)) in detriment of newborn astrocytes (in green) from the SVZ to the ischemic area (Figure 4(b)). DPCPX-treated MCAO mice showed a significant increase in DCX⁺ cells and mature neurons (NeuN⁺ cells) at day 28 post-ischemia in the brain area affected by the infarction (p < 0.05, Figures 4(c) and (d)). In contrast, MCAO induced a progressive significant increase in newborn astrocytes from days 8 to 28 after ischemia in agreement with our previous work,²¹ that was reverted by chronic DPCPX treatment (p < 0.01, Figure 4(e)). Similarly, a shift in the balance between neurogenesis and astrogliogenesis was observed in DPCPX-treated ischemic mice beginning at day 15 after ischemic brain injury (p < 0.001, Figures 4(f) and (g)). Furthermore, DCX⁺ cell density was higher at 15 and 28 days after stroke in the SVZ of ischemic mice treated with DPCPX (p < 0.01, Figures 4(h) and (i)). Finally, neuron maturity, as assessed by Sholl analysis, revealed an increase in both the number of intersections and the area occupied by neurites in ischemic mice treated with DPCPX at 28 days post-ischemia (p < 0.001, Figures 4(j) and (k). The maturity was also measured through double positive DCX/NeuN cells, where we observed an increase at 15 and 28 days (p < 0.05, Figures 4(l) and (m)). Hence, these results suggest that DPCPX treatment promotes neuronal production and maturity at the chronic phase of ischemic stroke.

Figure 4.

Chronic treatment with DPCPX increased new-born neurons following MCAO. Experimental design (a). Representative merged images of Doublecortin (DCX, pink) and Thrombospondin 4 (Thbs4, green) at 28 days after ischemic stroke in animals treated with either vehicle (top) or DPCPX (bottom) (b). The number of new-born neurons (DCX) (c), neurons (d) and new-born astrocytes (Thbs4) (e) were measured in the infarcted areas in both conditions at 8, 15 and 28 days after ischemia. The proportion of neurogenesis and astrogliogenesis in the infarcted area was measured at day 15 after MCAO (f, g). DCX expression in the SVZ of both groups 15 days post-stroke (h). DCX⁺ cells increase in the SVZ of ischemic mice treated with DPCPX (i). Representative images used for Sholl analysis in both groups (j). Sholl analysis results in ischemic mice treated with vehicle and DPCPX (k). Double positive cells DCX/NeuN were measured in the infarct regions (l). DCX/NeuN⁺ cells increased 15 and 28 days after ischemic stroke in mice treated with DPCPX (m). *p < 0.05, **p < 0.01 and ***p < 0.001. Scale bars = 500 µm (b), 10 µm (f) and 10 µm (h). Values are presented as scatter dot blot and bars (mean ± SD).

Chronic DPCPX treatment ameliorated motor and memory deficits after brain ischemia

The results above evidenced that chronic inhibition of A1AR receptor after brain ischemia by DPCPX treatment induces SVZ proliferation, differentiation into newborn neurons, and their subsequent maturation in the ischemic lesion. Next, we evaluated the impact of chronic DPCPX treatment on motor and cognitive decline following cerebral ischemia using a series of behavioral tests at different time points after MCAO. First, we assessed body weight, neurological score and lesion evolution following MCAO in DPCPX-treated animals compared to the vehicle group (Figure 5). Body weight showed no significant differences between the experimental groups (Figure 5(b)). However, ischemic mice treated with chronic DPCPX displayed an improvement on neurological outcomes (p < 0.001, Figure 5(c)) and survival rates (p < 0.001, Figure 5(d)) compared to ischemic animals treated with the vehicle. Despite this, the unusual mortality observed in the vehicle-treated MCAO group at 20 days post-MCAO might lead to an overestimation of the improved survival observed in DPCPX-treated ischemic mice. In addition, the infarct volume measured by cresyl violet staining was reduced in DPCPX-treated ischemic mice at 28 days post-ischemia (p < 0.05, Figures 5(e) and (f)). These results suggest that chronic DPCPX primarily promotes recovery at later stages of cerebral ischemia.

Figure 5.

Characterization of stroke outcome following treatment with DPCPX. Experimental design (a), body weight (b), neurological evaluation (c) and survival (d) were measured every day in sham and MCAO groups treated with either vehicle or DPCPX. Infarct volume was analyzed by cresyl violet staining (e). The infarct volume was reduced at day 28 after MCAO in DPCPX-treated mice (f). *p < 0.05 and ***p < 0.001. Scale bar = 2 mm (e). Values are presented as scatter dot blot (mean ± SD).

Subsequently, a set of behavioral tests were performed including sham conditions for both vehicle- and DPCPX-treated animals to monitor possible side-effects of the treatment (Figure 6). Motor deficits were measured by rotarod test at days 1, 8, 15, 21 and 28 post-MCAO (Figure 6(a)). No significant changes were observed in either sham group (vehicle/DPCPX) at any time point (Figure 6(b)). However, animals treated with DPCPX showed a faster recovery than vehicle-treated mice after MCAO, reaching sham control values within one week after the ischemic insult (p < 0.01, Figure 6(c)). Additionally, the pole test was carried out to assess the ability of mice to grasp and maneuver a pole, with assessments made daily from MCAO onset until euthanasia, restricted to ischemic animals (Figure 6(d)). Vehicle ischemic mice tended to fall rapidly, suggesting impaired skilled grip that was improved in DPCPX-treated mice after cerebral ischemia (Figure 6(e)). Motor symptoms were also assessed using the corner test (Figure 6(f)) which showed that ischemic mice tended to turn toward the infarcted hemisphere, as expected (Figure 6(g)). However, DPCPX-treated MCAO mice turned indifferently to both sides showing loss of laterality (p < 0.001, Figure 6(g)). Laterality was further evaluated using the cylinder test where ischemic mice treated with DPCPX showed similar values to sham controls one-week post-ischemia, and showed significant improvement compared to vehicle-treated mice at days 21 and 28 after brain ischemia (p < 0.05, Figure 6(h)). Finally, adhesive removal test showed that DPCPX-treated ischemic mice displayed similar latency values to those showed by sham animals and significantly decreased in comparison to vehicle-treated ischemic mice (p < 0.01, Figure 6(i)). These results suggest that chronic DPCPX treatment improves sensorimotor symptoms after ischemic stroke.

Figure 6.

Chronic DPCPX treatment improved motor and memory symptoms after MCAO. Experimental design for rotarod test (a). Latency to fall in sham (b) and MCAO (c) groups. Experimental design for pole test (d). Latency to fall was abnormal in ischemic mice treated with vehicle (e). Experimental design and scheme of the corner test (f). Corner test was evaluated in treated and sham mice at day 28 after MCAO (g). The number of impaired paws touching the cylinder and the time-to-remove the adhesive from the paw were evaluated with the cylinder test (h) and the adhesive removal test (i) at different time points and experimental conditions. Experimental design of the novel object recognition test (j). Time spent with the novel object (k–l) and discrimination index (m–n) was analyzed at 4 and 24 h (short-term). *p < 0.05, **p < 0.01 and ***p < 0.001. Values are presented as scatter dot blot and bars (mean ± SD).

To measure memory deficits, the novel object recognition test was performed at short-term (4 and 24 hours) after ischemia (Figure 6(j)). As expected, ischemic mice showed impaired short and long-term memory, as evidenced by both the time spent on the novel object (p < 0.05, p < 0.001, Figure 6(k) and (l)) and the discrimination index (p < 0.05, p < 0.001, Figure 6(m) and (n)). However, this impairment was almost restored when ischemic mice were treated with DPCPX, with both the time spent with the novel object and the discrimination index showing significant improvement (p < 0.05, p < 0.01, Figure 6(k) to (n)). These results suggest that DPCPX treatment protected against memory deficits induced by brain ischemia.

Discussion

A1ARs activation after ischemic stroke has traditionally been regarded as neuroprotective.^6,16 However, in this study we evidence the potential therapeutic role of A1ARs antagonists in ischemic stroke. The results prove that chronic blockage of A1ARs signaling after brain ischemia induces cellular proliferation, as evidenced by in vivo PET imaging and IHC. Notably, proliferation was pronounced in the SVZ, consistent with previous findings by Rueger and colleagues, who identified [¹⁸F]FLT as a radiotracer for imaging endogenous neural stem cells after brain ischemia in rats.^28,34 Additionally, we demonstrate using IHC that the proliferating cells induce the differentiation of new-born neurons in the ischemic areas of mice treated with the A1ARs antagonist DPCPX. Chronic treatment with DPCPX also facilitated the recovery of motor and memory deficits induced by brain ischemia. As a whole, these results suggest that blocking A1ARs signaling may serve as a potential therapeutic target for brain ischemia.

Previous studies have demonstrated that the timing of treatment initiation plays a critical role in the success of ischemic stroke recovery.³⁵ In particular, acute administration of A1ARs agonists following the onset of ischemia has been shown to be beneficial, suggesting that activation of this receptor, rather than its blockade, may represent a potential treatment for ischemic stroke.^14,15,36 Despite this, the effect of acute A1ARs agonists on stroke outcome remains controversial, with studies reporting both beneficial and detrimental impacts on ischemic damage.³⁷ A previous work by our team showed a worsening of ischemic stroke outcome after acute treatment with A1ARs antagonists, but a positive result with the use of agonists.³⁰

In our study, we demonstrated the beneficial effects of chronic A1ARs antagonist treatment in ischemic stroke, consistent with the findings of von Lubitz et al. who reported a negative effect when A1ARs antagonists were administered acutely, but a positive effect with chronic administration.³⁸ The chronicity of the treatment is beneficial and could be explained by compensatory mechanisms such as receptor hypersensitivity due to its prolonged inhibition. Indeed, we observed a non-significant increase of A1ARs expression in mice treated with DPCPX (Figures 2Sa–d), a phenomenon also described by others as protective in the progression of ischemic stroke.^35,39 While A1ARs upregulation may explain the long-term effects observed in our experiments, further studies are needed to elucidate whether short-term A1ARs blockage also provides benefits in ischemic stroke or could be effectively combined with other treatments. Additionally, other neural cells might participate in the ischemic cascade, potentially explaining the dual role of adenosine receptors following brain stroke.³⁰ We previously correlated [¹⁸F]FLT signal uptake to microglia/macrophages proliferation at 8 days after stroke,²⁹ along with A1AR overexpression in these cells that could also influence the effect of A1AR antagonists in ischemic mice.³⁰ This finding could also help explain, at least in part, the cellular source of the proliferative activity observed in the infarcted areas 8 days after stroke in our study. Therefore, future studies should also focus on cell-specific treatments to avoid side effects on the rest of the body and non-ischemic brain regions. Despite this controversy, we focused on how the blockage of A1ARs signaling affected neural stem cells in the SVZ after brain ischemia. Previously, we described the mechanisms underlying the production of neurons or glia, which are strongly linked to the adenosine activity.⁴⁰ The SVZ is closely interconnected with other brain regions, and it harbors an ultra-specialized microenvironment capable of detecting molecular changes in distant areas of the brain.⁴¹ Indeed, the SVZ is well-known for its response against brain stroke, mainly producing newborn astrocytes and migrating to the ischemic regions throughout the blood vessels.^20–23 We previously observed that SVZ cells mainly differentiated into astrocytes due to A1ARs activation and could be reverted when SVZ cells were treated with A1ARs antagonists.⁴⁰ This close relationship has also been observed by others particularly after stroke where metabolic disruption and adenosine accumulation occur.⁴² This suggests that SVZ cells differentiate into newborn astrocytes through A1ARs activation. Nevertheless, this signaling pathway may influence not only their differentiation but also the migration of these newborn astrocytes to the ischemic regions, enhancing blood vessel density. In addition, previous studies have observed that reactive microglia from the SVZ inhibit neuroblast production after stroke.^43,44 While these cell types are strongly influenced by A1ARs activity, further research is needed to elucidate whether A1AR inhibition limits microglial reactivity in SVZ, thereby promoting neurogenesis in ischemic areas, in contrast to untreated conditions.²¹

Our study demonstrates the induction of newborn neurons in ischemic regions after A1ARs inhibition by chronic DPCPX treatment, which is associated with an improvement in stroke prognosis. This supports the notion that therapeutic approaches aimed at replenishing damaged areas with newborn neurons, rather than newborn astrocytes, are crucial for improving stroke recovery and may serve as a potential strategy against brain ischemia. In conclusion, our results, based on in vivo PET imaging, behavioral tests and ex vivo IHC, provide valuable insights into how adenosine modulates SVZ cells and its impact on ischemic stroke.

Limitations

Future studies should consider conducting the same research experiments in both rats and mice due to the metabolic differences observed in different rodent species. Due to these differences, it would be necessary to adjust the dosage for both species, and therefore this aspect is a limitation of the present work. Moreover, a better understanding of the pathophysiology of ischemic stroke can be achieved by including animals of both sexes, as it is well known that the progression of stroke differs between males and females. Finally, the comorbidities associated with the disease should be studied in parallel with ischemic stroke. Ischemic stroke often occurs simultaneously with other medical conditions that could worsen or improve the stroke prognosis. Given their relevance, comorbidities must be incorporated into studies on ischemic stroke.

Supplemental Material

sj-pdf-1-jcb-10.1177_0271678X251345294 - Supplemental material for Chronic treatment with adenosine A1 receptor antagonist promotes neurogenesis and improves outcome after cerebral ischemia

Supplemental material, sj-pdf-1-jcb-10.1177_0271678X251345294 for Chronic treatment with adenosine A1 receptor antagonist promotes neurogenesis and improves outcome after cerebral ischemia by Maria Ardaya, Monica Benito-Muñoz, Esther Rubio-López, Maider Garbizu, Laura Aguado, Naroa Mocha-Muñoz, Leyre Iglesias, Unai Aldutzin, Carlos Matute, Federico N Soria, Vanessa Gómez-Vallejo, Aitzol García-Etxarri, Jordi Llop, Fabio Cavaliere and Abraham Martín in Journal of Cerebral Blood Flow & Metabolism

Footnotes

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was funded by grants from the ERA-NET NEURON under grant agreement N° CV_034, Spanish Ministry of Education and Science/FEDER RYC-2017- 22412, and PID2019-107989RB-I00, PID2022-138022OB-I00, PID2020-118546RB- I002, PID2023-148642OB-I00, PCI2022-134986-2 funded by MICIU/AEI/10.13039/501100011033 and European Union NextGenerationEU/PRTR, Instituto de Salud Carlos III (PI19/00936, co-funded by the European Regional Development Fund) and by the IKUR Strategy under the collaboration agreement between Ikerbasque Foundation and DIPC on behalf of the Department of Education of the Basque Government. A.G. E. acknowledges funding from the Programa de Ayuda de Apoyo a los agentes de la Red Vasca de Ciencia, Tecnología e Innovación acreditados en la categoría de Centros de Investigación Básica y de Excelencia (Programa BERC) from the Departamento de Universidades e Investigación del Gobierno Vasco and Centro Severo Ochoa AEI/CEX2018-000867-S from the Spanish Ministerio de Ciencia e Innovación. Finally, C.M. acknowledges funding from CIBERNED.

Acknowledgements

The authors would like to thank A. Lekuona and V. Salinas for technical support in the radiosynthesis. The authors also thank Juan Carlos Chara, Saioa Marcos and Zara Martínez for technical support.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Author contributions

M.A., M.B-M., E.R. and L.I. conducted experiments. M.A. collected and analyzed data. M.G., L.A., N.M. and U.A. helped to analyzed the PET imaging data. C.M., J.L.L, A.G.E. V.G.V and J.L.L performed the synthesis of the radiotracer and A.M. secured funding and provided infrastructural support. F.C. conceived the study. M.A. prepared the figures. M.A, F.N.S, J.L.L and A.M. wrote the paper.

Supplementary material

Supplemental material for this article is available online

ORCID iDs

Maria Ardaya

Carlos Matute

Abraham Martín

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