Lansoprazole Improves Long-Term Neurological Function via Alleviating Intestinal Injury in a Mouse Model of Intracerebral Hemorrhage

Summary

A collagenase-induced mouse model of intracerebral hemorrhage (ICH) was treated with lansoprazole. Lansoprazole not only alleviated ICH-caused intestinal injury and reduced levels of lipopolysaccharide (LPS) and IL-1β, but it also inhibited brain inflammation. Lansoprazole enhanced learning and memory (long-term neurological function) of ICH mice.

Abstract

Intracerebral hemorrhage (ICH) can compromise the gastrointestinal tract and trigger intestinal injury, contributing to poor clinical outcomes. This study investigated the effects of lansoprazole on intestinal injury and neurological function in mice with ICH. Mice were stereotactically injected with collagenase to establish an ICH model. Neurological function, brain water content, blood-brain barrier permeability, intestinal injury, and intestinal permeability were evaluated. Levels of LPS and IL-1β in the blood and brain were also assayed. Lansoprazole treatment was associated with alleviated intestinal injury, reduced levels of LPS and IL-1β in blood and brain, decreased brain water content, and inhibited brain inflammation in ICH mice. While lansoprazole administration did not lead to improved performance in the Garcia, forelimb placing, and rotarod tests (short-term neurological function), it significantly enhanced learning and memory (long-term neurological function). These findings indicated that lansoprazole treatment was associated with improved long-term neurological outcomes and reduced intestinal injury in ICH mice.

Introduction

Hemorrhagic stroke has high rates of mortality and disability. Recently, the prevalence of stroke in China has increased, making it the leading cause of death¹. Although less common, intracerebral hemorrhage (ICH) is markedly more lethal than ischemic stroke. Studies report a 40% mortality rate within 1 month of ICH; furthermore, approximately 75% of survivors are unable to live independently 1 year later².

The brain communicates with the gastrointestinal tract through several pathways, including the vagus nerve, enteric nervous system, and gut microbial metabolism. The brain-gut axis is a well-recognized and clinically relevant regulatory system³. The intestinal mucosa regulates the absorption of water, electrolytes, and nutrients, while preventing the entry of pathogens and toxins into the bloodstream. ICH can compromise this intestinal barrier and trigger gastrointestinal injury. Barrier dysfunction, in turn, contributes to malnutrition, immunosuppression, and poor clinical outcomes⁴. ICH-induced increases in intestinal permeability allow harmful endogenous substances to enter the bloodstream, triggering systemic inflammatory responses and multiple organ dysfunction syndrome. Damage to the intestinal mucosa following ICH can even allow gut bacteria to translocate into the bloodstream, potentially leading to systemic infections⁵. Many patients develop intestinal dysmotility, microbial dysbiosis, mucosal bleeding, and sepsis. One previous study showed that ICH caused the migration of intestinal pathogens and toxins to the brain⁶.

Lansoprazole, a proton pump inhibitor (PPI), irreversibly inhibits the proton pump and thereby reduces gastric acid secretion. It is used in the treatment of gastric and duodenal ulcers, erosive esophagitis, gastroesophageal reflux disease, and nonsteroidal anti-inflammatory drug-associated gastric ulcers⁷. A previous study indicated that PPI prophylaxis negatively affected patient outcomes in ischemic stroke⁸. Other published studies suggested an association between PPI use and cognitive decline. An epidemiological study showed that patients administered PPIs had an increased risk of developing dementia and Alzheimer's disease⁹. A prospective cohort study comparing long-term PPI users with non-users found that PPI use was associated with an increased risk of dementia, and the authors advised against PPI use in at-risk patients as a preventative measure¹⁰. The present study was performed to investigate the effects of lansoprazole on intestinal injury as well as short- and long-term neurological function (specifically learning and memory) in mice with ICH. We hypothesize that lansoprazole may inhibit the permeation of LPS by virtue of its gut-barrier-protective effects, alleviating systemic and neurological inflammation, and therefore improving the neurological function in ICH mice.

Protocol

The experiments were performed according to the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (NIH Publication No. 8023, revised 1978). The protocol was approved by the Ethics Committee of Yantai University (Approval No. YTDX20240526). Hazardous materials were disposed of according to institutional chemical waste protocols. Figure 1 illustrates a schematic diagram of the experimental design. The reagents and the equipment used are listed in the Table of Materials.

1. Animal details

Male CD-1 mice weighing 22-25 g were housed in a room with a 12-/12-h light/dark cycle. The animals were free to get food and water.

2. Preparation of the ICH mouse model

The mouse model of ICH was established according to a previous report¹¹. Briefly, mice were anesthetized with xylazine (10 mg/kg) and ketamine (90 mg/kg), then positioned in a stereotaxic frame. A small burr hole was drilled at the right coronal suture (2.2 mm lateral to the midline, 0.2 mm posterior to the bregma). A microsyringe was inserted into the right brain at a depth of 3.5 mm from the skull surface. Using a microinfusion pump, bacterial collagenase (0.075 U in 0.5 µL phosphate-buffered saline [PBS]) was infused at a rate of 0.15 µL/min. The needle was gently withdrawn after 5 min. Mice in the sham group received an equivalent volume of PBS instead of collagenase. Sealed the burr hole with bone wax, and the scalp incision was sutured. The body temperature of animals was maintained at 37°C until awakening.

3. Experimental design

Male CD-1 mice were randomly assigned to three groups: sham, model, and lansoprazole. The ICH model was established as described above. Thirty minutes after ICH induction, mice in the lansoprazole group received lansoprazole at a dose of 30 mg/kg (the dose of lansoprazole was selected according to a previous report¹²), administered once daily for three consecutive days. Mice in the sham and model groups received intraperitoneal injections of PBS. At 3 days post-ICH, short-term neurological function was assessed using the Garcia test, forelimb placing test, and rotarod test. On day 29 after ICH induction, the novel object recognition and water maze tests were conducted to evaluate long-term neurological function.

4. Garcia test

Two researchers, blinded to the group, performed the Garcia test according to a previously reported protocol¹³. Neurological function was assessed using a scoring system ranging from 0 to 21. The evaluation consists of seven subtests, each scored from 0 to 3 (0 = worst, 3 = best).

5. Forelimb placing test

Following the Garcia test, the forelimb placing test was performed according to a previously described method¹⁴. The mice were held in a vertical position, parallel to the tabletop, allowing their vibrissae to brush along the surface. Forelimb placement was observed over 10 consecutive trials. The percentage of left forelimb placement was calculated as: Left forelimb placement / (Left forelimb placement + Right forelimb placement) × 100%.

6. Rotarod test

After the forelimb placing test, the mice were placed on a rotating rod at 5 rpm for 3 min for training. The rod was accelerated from 5 rpm to 30 rpm over 4 min. The duration each mouse remained on the rod was recorded over a 5-min period, and the escape latency was recorded¹⁵.

7. Intestinal permeability and brain edema assay

After the rotarod test, six mice from each group were randomly selected. According to the previous method¹⁶, FITC-dextran was intragastrically administered at a dose of 300 mg/kg. Three hours later, the mice were anesthetized with isoflurane, and blood was collected from the retro-orbital venous plexus, centrifuged at 4 °C and 11,000 × g for 5 min. FITC-dextran content was assayed using a fluorescence plate reader at 485-nm excitation and 528-nm emission wavelengths. The animals were then decapitated, and the right hemisphere of the brain was collected and weighed to obtain the wet weight. The tissue was dried at 100 °C for 24 h to determine the dry weight. Brain water content (%) was calculated as: [(Wet weight − Dry weight) / Wet weight] × 100%.

8. Blood - brain barrier (BBB) permeability evaluation

After the rotarod test, six mice from each group were randomly selected for evaluating BBB permeability. A 2% Evans blue solution was intraperitoneally administered at 4 mL/kg. Three hours later, the mice were anesthetized with isoflurane, and transcardial perfusion was performed using PBS. The right hemisphere of the brain was homogenized in 50% trichloroacetic acid at a ratio of 3:1 (brain tissue: trichloroacetic acid). The homogenate was centrifuged at 15,000 × g and 4 °C for 20 min. Then, 500 µL of the supernatant was mixed with 500 µL of 70% ethanol and incubated overnight at 4 °C in the dark. The concentration of Evans blue was measured at 610 nm and quantified using a standard curve. Data are presented as µg of Evans blue per mg of brain tissue.

9. Histopathological examination of the intestine and brain

After the rotarod test, three mice from each group were randomly selected. The mice were anesthetized with isoflurane. The jejunum, ileum, and brain were collected and fixed in 10% formaldehyde. Tissues were sectioned at 5 µm thickness, and hematoxylin-eosin (HE) staining was performed. Histopathological examination of the intestine and brain was carried out using a microscope by two experimenters who were blinded to the group assignments. The number of microglia surrounding the hematoma was counted in representative sections. Cell counts were performed in five arbitrary visual fields across three sections per animal.

10. Measurement of LPS and IL-1β

After the rotarod test, six mice from each group were randomly selected. The mice were anesthetized with isoflurane, and blood was collected. Blood samples were centrifuged at 5,000 × g and 4 °C for 10 min, and serum was collected. The right hemisphere of the brain was harvested, and brain homogenates were prepared at a ratio of 1:3 (brain weight in grams to PBS volume in milliliters). The homogenates were centrifuged at 12,000 × g and 4 °C for 10 min. Protein concentrations in the supernatant were determined using a BCA kit. Levels of LPS and IL-1β in both serum and brain homogenate were measured using ELISA kits, following the manufacturer's instructions.

11. Transmission electron microscopy

For electron microscopic examination, three animals from each group were randomly selected after the rotarod test. The mice were anesthetized with isoflurane and underwent transcardiac perfusion with 2% glutaraldehyde and 2.5% paraformaldehyde in 0.1 M PBS (following institutionally approved protocols). The brain, jejunum, and ileum were then harvested and placed in 2% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) at 4 °C overnight. The samples were subsequently transferred to 1% osmium tetroxide for 1 h at room temperature. Tissues were cut into ultrathin sections (50 nm). After dehydration, the sections were stained with uranyl acetate and lead citrate for observation under a transmission electron microscope. All sections were evaluated in a blinded manner and photographed using a transmission electron microscope.

12. Novel object recognition

At 29 days after ICH modeling, the novel object recognition test was performed to assess the learning and memory function of mice. On the first day, no objects were placed in the apparatus (length: 40 cm, width: 40 cm, height: 40 cm), allowing the mice to explore freely for 5 min. On the second day, two identical objects were placed in the apparatus, and the mice were allowed to explore them for 5 min. Twenty-four hours later, one of the objects was replaced with a novel object. The mice explored the two objects for 5 min again. Exploration time was recorded when the mouse's nose touched an object or when its head was oriented toward the object within a 1-cm distance. The time spent exploring the novel object was measured and calculated. A longer exploration time of the novel object indicates better learning and memory ability.

13. Morris water maze

At 32 days after ICH modeling, learning and memory functions were assessed using the Morris water maze. The apparatus consisted of a circular pool (diameter: 100 cm, height: 40 cm) filled with water (depth: 25 cm) maintained at 22 °C ± 1 °C. An escape platform (diameter: 9 cm) was submerged 1 cm below the surface of the water. In the navigation test, mice underwent four trials per day. The time taken to find the platform was recorded as the escape latency. Mice that failed to locate the platform within 60 s were gently guided to it. On the fifth day, a 60-s probe test was conducted. Memory function was evaluated by recording the escape latency, number of crossings of the platform, and time spent in the target quadrant. Data were analyzed using Video Tracking Software.

14. Statistical analysis

Data were analyzed using GraphPad Prism software version 7.0. Results are presented as mean ± standard deviation. The Shapiro-Wilk test was used to assess whether data from each group followed a normal distribution. Differences among groups were analyzed by one-way ANOVA followed by Tukey's post hoc test. For non-parametric comparisons, the Kruskal-Wallis test followed by Dunn's test was used. A p-value of <0.05 was considered statistically significant.

Results

Effects of lansoprazole on short-term neurological function in ICH mice

Compared with the sham group, mice in the ICH group showed significantly lower scores in the Garcia test (p < 0.01). However, mice in the lansoprazole group did not show improved Garcia scores compared with the ICH group (p > 0.05) (Figure 2A). Similarly, the percentage of left forelimb p...

Discussion

Intestinal injury is a common complication in patients with ICH. ICH-induced intestinal damage can lead to malnutrition, prolonged hospitalization, poor outcomes, and even mortality¹⁷. Using an ICH animal model, this study demonstrated for the first time that lansoprazole attenuates intestinal injury in ICH mice. Although lansoprazole did not show beneficial effects on short-term neurological function, it significantly improved long-term neurological outcomes.

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Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
BCA kit	Beyotime Biotechnology	P0009
Collagenase	Sigma-Aldrich	128M4107v
Evans blue dye	Sigma-Aldrich	MKCG7507
FITC-dextran	Sigma-Aldrich	46944
GraphPad Prism 7.0	GraphPad statistical Software, Boston, MA, USA, www.graphpad.com	Prism 7.0
Lansoprazole	Shandong Yuxin pharmaceutical Co., Ltd.	524093053
LPS ELISA kit	Cusabio biotech Co., Ltd.	M25036407
Mice	Jinan Pengyue Experimental Animal Breeding Co., Ltd.	SCXK20220006
Microinfusion pump	World Precision Instruments	Micro 3
Microsyringe	Hamilton Company	SYR 10 μL
Olympus microscope	Olympus Co., Ltd.	IX-70
Rotarod	Panlab Co., Ltd.	LE8205
Stereotaxic frame	Kopf Instruments	Model 902-A
TNF-α ELISA kit	Beyotime Biotechnology	PI301
Transmission electron microscope	JEOL Ltd.	JEM-100SX
Video Tracking Software	Panlab Co., Ltd.	Smart 3.0