Sex-dependent effects of ultra-low-dose-THC preventive treatment on neuroinflammation and cognitive decline in 5xFAD mice methods
Aim. Evidence-backed execution summary for Sex-dependent effects of ultra-low-dose-THC preventive treatment on neuroinflammation and cognitive decline in 5xFAD mice methods from Sex-dependent effects of ultra-low-dose-THC preventive treatment on neuroinflammation and cognitive decline in 5xFAD mice.
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mouse
Subject model for the experiment.
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- confirm full cohort details in the source paper
Main text
reagent used in the protocol.
- Use
- Although the mechanism of AD has been investigated for more than 50 years, currently, there are only a few available drugs for AD that can slow Aβ accumulation and treat some of the cognitive and emotional symptoms. In the past two to three years, new drugs have been approved for Alzheimer's disease, incl...
Main text
reagent used in the protocol.
- Use
- In the last decade, studies have found many changes in the endocannabinoid system in AD [ - ], yielding several clinical [ - ] and pre-clinical [ - ] studies on the benefits of cannabis and its main compound - 9Δ-tetrahydrocannabinol (THC). However, the use of THC in patients is hi...
Main text
reagent used in the protocol.
- Use
- One of the known effects of THC treatment is the modulation of the immune system and reduction of neuroinflammation [ ]. This can be important in AD, as the accumulation of Aβ exacerbates neuroinflammation in the AD brain [ ], raising the levels of pro-inflammatory cytokines [ ]. This chronic neuroinflammation...
Treatments
reagent used in the protocol.
- Use
- Δ9-THC (donated by Prof. Mechoulam, the Hebrew University, Jerusalem and by NIDA, USA) was dissolved from a stock solution in ethanol into a vehicle solution consisting of 1:1:18 ethanol: cremophor (Sigma-Aldrich): saline and was administered i.p at a dose of 0.002 mg/kg.
Behavioural tests
reagent used in the protocol.
- Use
- Learning ability and spatial memory were evaluated using MWM. This classical spatial learning assay was performed as previously described [ ]. Briefly, the mouse was introduced into a round pool at different starting points, spatial cues (such as a black cross on the white wall, etc.) were visible for the mice, and...
Y maze (YM)
reagent used in the protocol.
- Use
- Place recognition test (PLT): This assay tests long-term memory and utilizes the natural tendency of mice to explore novel stimuli. It was performed as previously described [ ]. Animals were placed in a familiar 40 cm × 40 cm × 40 cm open field box, with two objects (6*5*7 cm cube. A pi...
Y maze (YM)
reagent used in the protocol.
- Use
- RT-PCR: After the last behavioral test, mice were euthanized using cervical dislocation, and the hippocampus and PFC tissue were removed and kept at -80c. Total RNA was extracted from the hippocampus using the PureLink RNA Mini Kit (Rhenium, Israel) according to the manufacturer's instructions. SYBR Gree...
Preventive treatment with ULD-THC modifies hippocampal-neuroinflammation in male 5xFAD mice, with no effect on PFC-ne...
reagent used in the protocol.
- Use
- Next, we examined tissue inhibitor of metalloproteinase 3 (TIMP3) (Fig. C). TIMP-3 plays a complex role in AD and aging. On the one hand, it inhibits Matrix metalloproteinases (MMPs)-induced inflammation, has a neuroprotective effect [ ] and is associated with protection against neuroinflammation [ ]. On the o...
Main text
Alzheimer's disease (AD) is the most common cause of dementia, with women being twice as likely to be affected compared to men. AD patients suffer from memory loss, poor judgment, confusion and neuropsychiatric disorders, accompanied by the accumulation of beta-amyloid (Aβ) and tau tangles in the brain [...
- Use
- Alzheimer's disease (AD) is the most common cause of dementia, with women being twice as likely to be affected compared to men. AD patients suffer from memory loss, poor judgment, confusion and neuropsychiatric disorders, accompanied by the accumulation of beta-amyloid (Aβ) and tau tangles in the brain [...
Main text
In the last decade, studies have found many changes in the endocannabinoid system in AD [ - ], yielding several clinical [ - ] and pre-clinical [ - ] studies on the benefits of cannabis and its main compound - 9Δ-tetrahydrocannabinol (THC). However, the use of THC in patients is hi...
- Use
- In the last decade, studies have found many changes in the endocannabinoid system in AD [ - ], yielding several clinical [ - ] and pre-clinical [ - ] studies on the benefits of cannabis and its main compound - 9Δ-tetrahydrocannabinol (THC). However, the use of THC in patients is hi...
Main text
One of the known effects of THC treatment is the modulation of the immune system and reduction of neuroinflammation [ ]. This can be important in AD, as the accumulation of Aβ exacerbates neuroinflammation in the AD brain [ ], raising the levels of pro-inflammatory cytokines [ ]. This chronic neuroinflammation...
- Use
- One of the known effects of THC treatment is the modulation of the immune system and reduction of neuroinflammation [ ]. This can be important in AD, as the accumulation of Aβ exacerbates neuroinflammation in the AD brain [ ], raising the levels of pro-inflammatory cytokines [ ]. This chronic neuroinflammation...
Behavioural tests
Mice were tested for two weeks in the following order: in the first week, the mice were tested first with the Open field habituation (OFH), then PLT (Place recognition test) and lastly Y-MAZE. In the second week, mice were tested in the Morris water maze (MWM.)
- Use
- Mice were tested for two weeks in the following order: in the first week, the mice were tested first with the Open field habituation (OFH), then PLT (Place recognition test) and lastly Y-MAZE. In the second week, mice were tested in the Morris water maze (MWM.)
Y maze (YM)
This test evaluated short-term working memory (PFC-dependent task) and was performed as previously described [, ]. Briefly, the Y-maze is a three-arm maze with all arms at equal angles, 30 cm in length and 5 cm in width with walls 12 cm high. Mice were initially placed in the middle, and the sequence...
- Use
- This test evaluated short-term working memory (PFC-dependent task) and was performed as previously described [, ]. Briefly, the Y-maze is a three-arm maze with all arms at equal angles, 30 cm in length and 5 cm in width with walls 12 cm high. Mice were initially placed in the middle, and the sequence...
Y maze (YM)
Place recognition test (PLT): This assay tests long-term memory and utilizes the natural tendency of mice to explore novel stimuli. It was performed as previously described [ ]. Animals were placed in a familiar 40 cm × 40 cm × 40 cm open field box, with two objects (6*5*7 cm cube. A pi...
- Use
- Place recognition test (PLT): This assay tests long-term memory and utilizes the natural tendency of mice to explore novel stimuli. It was performed as previously described [ ]. Animals were placed in a familiar 40 cm × 40 cm × 40 cm open field box, with two objects (6*5*7 cm cube. A pi...
Y maze (YM)
Open-field habituation (OFH): This test evaluates long-term non-associative, non-aversive spatial learning by measuring the decrease in the exploratory activity of the animal in a test session carried out 24 h after the first exploration session (delta of 2nd session - 1 st session) [ ]. In Brief, a...
- Use
- Open-field habituation (OFH): This test evaluates long-term non-associative, non-aversive spatial learning by measuring the decrease in the exploratory activity of the animal in a test session carried out 24 h after the first exploration session (delta of 2nd session - 1 st session) [ ]. In Brief, a...
Y maze (YM)
The arenas of the Y-maze, OFH and PLT were cleaned with Alcohol between mice, in order to eliminate olfactory cues. All the behavioral assays were photographed using a video camera, and data were recorded and analyzed using the 13th Noldus EthoVision software (tracking the center-mass in MWM, Y-maze and OFH, and nos...
- Use
- The arenas of the Y-maze, OFH and PLT were cleaned with Alcohol between mice, in order to eliminate olfactory cues. All the behavioral assays were photographed using a video camera, and data were recorded and analyzed using the 13th Noldus EthoVision software (tracking the center-mass in MWM, Y-maze and OFH, and nos...
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Main text
Although the mechanism of AD has been investigated for more than 50 years, currently, there are only a few available drugs for AD that can slow Aβ accumulation and treat some of the cognitive and emotional symptoms. In the past two to three years, new drugs have been approved for Alzheimer's disease, including symptomatic treatments like Benzgalantamine (a cholinesterase inhibitor, FDA-approved in July 2024) and disease-modifying therapies like Lecanemab and Donanemab (anti-amyloid monoclonal antibodies, FDA-approved in July 2023 and July 2024, respectively) [ ]. While these therapies mark significant progress, they do not prevent the disease. The distinction between slowing progression and true prevention is crucial, as no existing treatment can halt the pathological processes before they begin. Thus, while there have been advances in treating AD, there is still no way to...
Main text
In the last decade, studies have found many changes in the endocannabinoid system in AD [ - ], yielding several clinical [ - ] and pre-clinical [ - ] studies on the benefits of cannabis and its main compound - 9Δ-tetrahydrocannabinol (THC). However, the use of THC in patients is hindered by the harmful effects of high doses of chronic THC use [ ]. One strategy that has been suggested to tackle this issue is to use an ultra-low dose of THC (ULD-THC) [ - ]. Indeed, we have recently shown that a single dose of ULD-THC has a beneficial effect on age-related cognitive decline in old female mice [ ] and alleviated AD-related cognitive impairments in the 5xFAD AD mice model [ ]. However, the potential of cannabinoids in the prevention of AD is still unexplored. The primary objective of this study was to evaluate the effects of preventive treatment of...
Main text
Building on prior findings of memory improvement following ULD-THC treatment after disease onset [ ], this study employs a preventive regimen to explore sex-specific molecular and behavioral responses to ULD-THC administered at earlier stages of AD pathology. We investigate here the preventive effects of ULD-THC on neuroinflammation and cognitive deficits in 5xFAD male and female mice and explore the molecular changes in both the hippocampus and PFC. 5xFAD mice start to exhibit early brain pathologies between 1.5 and 3 months, with behavioural phenotype staring at 6 months [, ]. Give our previous experiment indicate a beneficial effect of ULD-THC for up to 3-4 weeks after the single treatment, we employed a monthly ULD-THC treatments from 2 to 5 months of age were followed by behavioral assessments and molecular analyses at 6 months.
Study design
To determine the prevention efficacy of ULD-THC in the 5xFAD mice model, 80 3-month-old male and female 5xFAD mice and their WT littermates were treated with three i.p. injections of either ULD-THC or vehicle (10 per group) once a month for 3 months from the age of 3 to 5 months. At the age of 6 months, 3 weeks after the last treatment, mice were tested for both short-term and long-term spatial memory. The mice underwent one test per day for 2 weeks, after which they were sacrificed for biochemical examination. All male and female mice were subjected to the same behavioral and biochemical tests following an identical timeline.
Treatments
Δ9-THC (donated by Prof. Mechoulam, the Hebrew University, Jerusalem and by NIDA, USA) was dissolved from a stock solution in ethanol into a vehicle solution consisting of 1:1:18 ethanol: cremophor (Sigma-Aldrich): saline and was administered i.p at a dose of 0.002 mg/kg.
Treatments
Mice were treated with a monthly i.p. injection of either ULD-THC or vehicle, starting before disease onset at the age of 3-5 months. Thus, each mouse was treated 3 times - once at the age of 3 months, once at the age of 4 months and once at the age of 5 months. At the age of 6 months, 3 weeks after the last treatment, the animals were assessed for cognitive deficits.
Behavioural tests
Learning ability and spatial memory were evaluated using MWM. This classical spatial learning assay was performed as previously described [ ]. Briefly, the mouse was introduced into a round pool at different starting points, spatial cues (such as a black cross on the white wall, etc.) were visible for the mice, and was allowed 60 s to find the platform. The water was kept at a temperature of 23 degrees Celsius. Each mouse swam four times per day for three consecutive days. The time and route required for the mouse to find the immersed platform was recorded. On day 4 of the assay, the platform was removed, and the mouse was allowed to swim for 60 s ("probe test"). The time the mice spent in the platform zone (the location of the missing platform) was recorded.
Measurement outputs
What raw and processed outputs should exist?
5xFAD male mice were bred with C57B/Rcc females, and offspring were genotyped by PCR analysis of tail DNA to identify the tg-mice. Non-Tg littermates were used as control WT mic...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
In hippocampal astrocytes (Fig. A), we saw a significant difference between the groups [F (3, 36) = 32.98, < 0.0001], with both non-treated AD mice and t...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Lastly, we examined SGK-1 expression, which is known to regulate microglia and facilitate neuroprotection [, ]. In the Hippocampus (Fig. C), one-way ANOVA yielded a signi...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
One of the known effects of THC treatment is the modulation of the immune system and reduction of neuroinflammation [ ]. This can be important in AD, as the accumulation of A...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Analysis plan
How should the outputs become interpretable results?
Acquisition
Collect raw experimental outputs with enough metadata to preserve sample identity, condition, and timing.
inferred from protocolPreprocessing / cleaning
In hippocampal astrocytes (Fig. A), we saw a significant difference between the groups [F (3, 36) = 32.98, < 0.0001], with both non-treated AD mice and treated-AD mice exhibiting elevated expression compared to vehicle-treated WT and THC-treated WT (<&#...
from paperScoring or quantification
Quantify the primary readouts for this experiment: 5xFAD male mice were bred with C57B/Rcc females, and offspring were genotyped by PCR analysis of tail DNA to identify the tg-mice. Non-Tg littermates were used as control WT mic...; In hippocampal astrocytes (Fig. A), we saw a significant difference between the groups [F (3, 36) = 32.98, < 0.0001], with both non-treated AD mice and t...; Lastly, we examined SGK-1 expression, which is known to regulate microglia and facilitate neuroprotection [, ]. In the Hippocampus (Fig. C), one-way ANOVA yielded a signi...; One of the known effects of THC treatment is the modulation of the immune system and reduction of neuroinflammation [ ]. This can be important in AD, as the accumulation of A....
from paperStatistical comparison
In hippocampal astrocytes (Fig. A), we saw a significant difference between the groups [F (3, 36) = 32.98, < 0.0001], with both non-treated AD mice and t...; Lastly, we examined SGK-1 expression, which is known to regulate microglia and facilitate neuroprotection [, ]. In the Hippocampus (Fig. C), one-way ANOVA yielded a signi...; Male and female 5xFAD mice received monthly ULD-THC injections from 3 to 5 months of age, before significant pathology emerged. At 6 months, behavioral assessments were conducte...; Although the mechanism of AD has been investigated for more than 50 years, currently, there are only a few available drugs for AD that can slow Aβ accumulation and treat so...
from paperReporting output
Report representative outputs alongside summary comparisons for 5xFAD male mice were bred with C57B/Rcc females, and offspring were genotyped by PCR analysis of tail DNA to identify the tg-mice. Non-Tg littermates were used as control WT mic..., In hippocampal astrocytes (Fig. A), we saw a significant difference between the groups [F (3, 36) = 32.98, < 0.0001], with both non-treated AD mice and t..., Lastly, we examined SGK-1 expression, which is known to regulate microglia and facilitate neuroprotection [, ]. In the Hippocampus (Fig. C), one-way ANOVA yielded a signi..., One of the known effects of THC treatment is the modulation of the immune system and reduction of neuroinflammation [ ]. This can be important in AD, as the accumulation of A....
inferred from protocolStructured statistical methods
In hippocampal astrocytes (Fig. A), we saw a significant difference between the groups [F (3, 36) = 32.98, < 0.0001], with both non-treated AD mice and t...; Lastly, we examined SGK-1 expression, which is known to regulate microglia and facilitate neuroprotection [, ]. In the Hippocampus (Fig. C), one-way ANOVA yielded a signi...; Male and female 5xFAD mice received monthly ULD-THC injections from 3 to 5 months of age, before significant pathology emerged. At 6 months, behavioral assessments were conducte...; Although the mechanism of AD has been investigated for more than 50 years, currently, there are only a few available drugs for AD that can slow Aβ accumulation and treat so...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (7)
Although the mechanism of AD has been investigated for more than 50 years, currently, there are only a few available drugs for AD that can slow Aβ accumulation and treat some of the cognitive and emotional symptoms. In the past two to three years, new drugs have been approved for Alzheimer's disease, including symptomatic treatments like Benzgalantamine (a cholinesterase inhibitor, FDA-approved in July 2024) and disease-modifying therapies like Lecanemab and Donanemab (anti-amyloid monoclonal antibodies, FDA-approved in July 2023 and July 2024, respectively) [ ]. While these therapies mark significant progress, they do not prevent the disease. The distinction between slowing progression and true prevention is crucial, as no existing treatment can halt the pathological processes before they begin. Thus, while there have been advances in treating AD, there is still no way to prevent the disease [ ].
In the last decade, studies have found many changes in the endocannabinoid system in AD [ - ], yielding several clinical [ - ] and pre-clinical [ - ] studies on the benefits of cannabis and its main compound - 9Δ-tetrahydrocannabinol (THC). However, the use of THC in patients is hindered by the harmful effects of high doses of chronic THC use [ ]. One strategy that has been suggested to tackle this issue is to use an ultra-low dose of THC (ULD-THC) [ - ]. Indeed, we have recently shown that a single dose of ULD-THC has a beneficial effect on age-related cognitive decline in old female mice [ ] and alleviated AD-related cognitive impairments in the 5xFAD AD mice model [ ]. However, the potential of cannabinoids in the prevention of AD is still unexplored. The primary objective of this study was to evaluate the effects of preventive treatment of ULD-THC on neuroinflammation and cognitive deficits in male and female 5xFAD AD mice.
Building on prior findings of memory improvement following ULD-THC treatment after disease onset [ ], this study employs a preventive regimen to explore sex-specific molecular and behavioral responses to ULD-THC administered at earlier stages of AD pathology. We investigate here the preventive effects of ULD-THC on neuroinflammation and cognitive deficits in 5xFAD male and female mice and explore the molecular changes in both the hippocampus and PFC. 5xFAD mice start to exhibit early brain pathologies between 1.5 and 3 months, with behavioural phenotype staring at 6 months [, ]. Give our previous experiment indicate a beneficial effect of ULD-THC for up to 3-4 weeks after the single treatment, we employed a monthly ULD-THC treatments from 2 to 5 months of age were followed by behavioral assessments and molecular analyses at 6 months.
To determine the prevention efficacy of ULD-THC in the 5xFAD mice model, 80 3-month-old male and female 5xFAD mice and their WT littermates were treated with three i.p. injections of either ULD-THC or vehicle (10 per group) once a month for 3 months from the age of 3 to 5 months. At the age of 6 months, 3 weeks after the last treatment, mice were tested for both short-term and long-term spatial memory. The mice underwent one test per day for 2 weeks, after which they were sacrificed for biochemical examination. All male and female mice were subjected to the same behavioral and biochemical tests following an identical timeline.
Δ9-THC (donated by Prof. Mechoulam, the Hebrew University, Jerusalem and by NIDA, USA) was dissolved from a stock solution in ethanol into a vehicle solution consisting of 1:1:18 ethanol: cremophor (Sigma-Aldrich): saline and was administered i.p at a dose of 0.002 mg/kg.
Mice were treated with a monthly i.p. injection of either ULD-THC or vehicle, starting before disease onset at the age of 3-5 months. Thus, each mouse was treated 3 times - once at the age of 3 months, once at the age of 4 months and once at the age of 5 months. At the age of 6 months, 3 weeks after the last treatment, the animals were assessed for cognitive deficits.
Learning ability and spatial memory were evaluated using MWM. This classical spatial learning assay was performed as previously described [ ]. Briefly, the mouse was introduced into a round pool at different starting points, spatial cues (such as a black cross on the white wall, etc.) were visible for the mice, and was allowed 60 s to find the platform. The water was kept at a temperature of 23 degrees Celsius. Each mouse swam four times per day for three consecutive days. The time and route required for the mouse to find the immersed platform was recorded. On day 4 of the assay, the platform was removed, and the mouse was allowed to swim for 60 s ("probe test"). The time the mice spent in the platform zone (the location of the missing platform) was recorded.
Machine-readable layer
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"item": "https://replicatescience.com/experiments/sex-dependent-effects-of-ultra-low-dose-thc-preventive-treatment-on-neuroinflammation-and-cognitive-decline-in-5xfad-mice-methods-keren-nitzan-pmc12866039/sex-dependent-effects-of-ultra-low-dose-thc-preventive-treatment-on-neuroinflamm"
}
]
}
]