As a leading CRO for CNS drug development, QPS Neuropharmacology provides induced animal models of several neurodegenerative and rare diseases. Animal models that recapitulate some pathophysiological and behavioral characteristics of human disorders can be used to develop new disease-specific drugs, screen compounds for lesion efficacy or toxicity, or study gene function. Furthermore, QPS Neuropharmacology offers virus injections of different types to induce a plethora of different pathologies by introducing cDNA or shRNA of your target protein.
Special Diet
People’s diet can strongly influence their health and wellbeing. Certain diet ingredients can even affect the onset and progression of some diseases. Feeding mice with a special diet can therefore be used to analyze the effect of a diet or dietary component on typical disease pathologies. Depending on the genetic background of mice used for the feeding study, the diet can be utilized to induce for example features of the metabolic syndrome such as obesity and insulin resistance or atherosclerosis but also to affect the pathology of Alzheimer’s or Parkinson’s disease transgenic mice.
Wild type or transgenic mice are fed with the diet of your choice – for instance with a Western-type, high/low fat, or high fructose/sucrose diet. Animals can be analyzed depending on your research question for the following readouts:
In vivo:
- Body weight
- Food consumption (caloric intake or consumed dietary components such as fat)
- In vivo blood sampling
- Gut transit time
- Glucose tolerance
- Activity and motor abilities
- Nesting Behavior
- Cognition
- BBB permeability by Evans Blue test (terminal)
Ex vivo:
- Preparation and analysis of different tissues, including various fat or gastrointestinal tissues
- Inflammation marker levels (cytokines) by MesoScale Discovery
- Serum lipid profile (LDL, HDL, total cholesterol, triglycerides)
- Disease-specific markers (Aβ, tau, p-tau, α-synclein)
- Preparation of Swiss roll of intestines and further analyses for
- PGP9.5 to mark enteric neurons
- Vimentine to mark mesenchymal cells including fibroblasts Leukocyte infiltration
- B- and T-cell marker (B220/CD45R, CD3)
- Monocytes/Macrophages (F4/80, CD68, CD11b, Iba1)
Figure 1: Immunofluorescent and H&E labeling of the colon of a wild type mouse fed with Western-type diet. Intestines were prepared as Swiss roll and labeled with PGP9.5 to visualize enteric neurons and vimentine to mark mesenchymal cells including fibroblast in the colon. Sections were counterstained with DAPI for visualization of cell nuclei. AF: Autofluorescence. Scale bar: 100 µm. H&E staining of the colon proving a structural overview of the tissue.
Virus Injections
Tau Seeding
Biosafety level 2 experiments are becoming more and more indispensable in the field of neurodegenerative and rare disease research. With this method, target proteins can be expressed or suppressed in distinct brain regions by intracerebral injection or more systemically by intraventricular or even intravenous injection of virus expressing cDNA or shRNA of the target protein.
The fully AAALAC accredited animal facility of QPS Neuropharmacology maintains a Biosafety Level 2 (BSL2) laboratory for mouse and rat experiments, including but not limited to e.g. Lenti-, Adeno- and Adeno-associated (AAV) virus injections.
To analyze the effect of specific viruses for the induction or treatment of Alzheimer’s or other cognitive diseases, injections in the mouse ERC can be performed.
In the here presented seeding study, P301L tau AAV was injected in the ERC of APPSL mice and spreading of tau in the hippocampus could be observed.
Figure 1: Recombinant Adeno-associated virus serotype 9 (AAV9) with human P301L Tau gene or empty vector was intracerebrally injected into the ERC of 3 month old male APPSL mice. A: Verification of injection site by ink injection into the entorhinal cortex. B: Tau expression in the ERC after P301L Tau virus injection. C: Spreading of tau into the hippocampus after P301L tau injection into the ERC of APPSL mice. All analyses were performed 1 month after virus injection.
Alzheimer’s Disease
Scopolamine-treated Mice
Scopolamine is a tropane alkaloid drug with competitive antagonism at muscarinic acetylcholine receptors (mAChR). Systemic application of scopolamine disrupts the performance in several reference memory tasks, such as object discrimination, radial arm maze, water maze and fear conditioning. The scopolamine-induced cognitive impairment can be reversed by cholinesterase inhibitors. Therefore, this model can be used to mimic cognitive dysfunction observed in dementia and AD and is a useful initial screening method to identify therapeutic candidates.
Figure 1: Effect of 1.0 mg/kg Scopolamine (Scop) on Passive Avoidance response of Wistar rats. Latency to enter the dark compartment. Effect of Scopolamine can be reduced by 1.0 mg/kg methylphenidate treatment. Mean ± SEM; Kruskal-Wallis test with Dunn`s multiple comparison test. n = 12 per group; **p<0.01.
Parkinson’s Disease
MPTP Mouse Model
Mice that receive acute, chronic or subchronic administration of the pyridine toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) selectively lose significant numbers of dopaminergic neurons in two midbrain structures, the substantia nigra (SN) and the ventral tegmental area (VTA). Loss of dopamine cells in the SN mimics the clinical condition of Parkinson’s disease and leads to motor dysfunction.
The dopaminergic loss in mouse VTA is of unknown relevance to Parkinson’s disease but may contribute to the cognitive deficits of Parkinson’s disease because of these neurons’ projections to the frontal cortex.
MPTP-treated mice are a suitable model to study motor deficits and the loss of dopaminergic neurons as well as possible influences of drugs on these parameters.
Figure 1: Open Field test. Animals were injected with 4 x 20 mg/kg MPTP or vehicle at one day. Two days after treatment, animals were tested in the Open field test for activity. n = 9-10 per group; unpaired t-test; Mean + SEM. ***p<0.001.
Figure 2. Tyrosine hydroxylase (TH) quantification in the substantia nigra after MPTP lesion. Animals were injected with 4 x 20 mg/kg MPTP or vehicle at one day. Six days after treatment, animals were sacrificed and the substantia nigra analyzed for TH levels. n = 3-5 per group; unpaired t-test; Mean + SEM. *p<0.05.
6-OHDA Lesion Model
Unilateral local application of 6-hydroxydopamine (6-OHDA), a neurotoxic substance that preferentially affects catecholaminergic neurons, is a well-established model for analyzing effects of loss of dopaminergic neurons in the substantia nigra pars compacta and their major target area, the dorsal striatum.
The advantage of unilateral lesions is two fold, I. the contralateral hemisphere serves as an internal control that facilitates comparison of lesion effects between individuals and experimental groups during histological analysis, and II. lesion efficacy in individual animals can be estimated prior to sacrifice using certain behavioral tests.
Figure 1. Coronal sections of 6-OHDA-injected mice. Tyrosine hydroxylase (TH) immunofluorescent labeling (upper picture). GFAP and TH immunofluorescent labeling to visualize astrocytosis (lower picture). Cpu: Caudate-Putamen; VTA: ventral tegmental area; SNpc: substantia nigra pars compacta. Arrows indicate injection canal.
Figure 2: Analysis of mice in the corridor test revealed that 6-OHDA injected mice preferred the right corridor side over the left while sham injected mice had no preference. n = 16-30; Mean + SEM. Two way ANOVA with Bonferroni’s post hoc test. ***p<0.001.
Haloperidol Induced Catalepsy
Catalepsy is a neuronal condition that can be observed in Parkinson’s disease, epilepsy, catatonia but also as adverse reaction to prescribed medications e.g. against schizophrenia. It is characterized by seizures with a loss of sensation and consciousness accompanied by rigidity of the body. By treating rats with the dopamine D2 receptor antagonist haloperidol, catalepsy can be acutely induced for several hours, mimicking the typical symptoms like loss of consciousness and rigidity.
Sprague Dawley rats were subcutaneously injected with 1 mg/kg Haloperidol. After 30 minutes, catalepsy was measured and additionally, animals showing a full catalepsy were treated orally with the positive compound or vehicle. Animals were retested for catalepsy at several time points after drug administration.
Catalepsy was evaluated by gently placing the front limbs of each rat over an 8 cm high horizontal bar; catalepsy was measured as time animals spent motionless. A cut-off time of 120 seconds was used.
Figure 1: Haloperidol-induced Catalepsy after treatment with a positive compound: Time animals spent rigid after s.c. treatment with 1 mg/kg haloperidol, followed by oral treatment with a positive compound. Animals were repeatedly tested for catalepsy. n = 10. Mean ± SEM. Two-way ANOVA with Bonferroni’s repeated measure post hoc test. ***p<0.001. T.I.: Test item / positive compound.
Gaucher Disease
Conduritol-B-Epoxide (CBE)-Induced Gaucher Disease
Gaucher disease (GD) is the most common lysosomal storage disorder. The main pathological hallmark is the intracellular accumulation of glucosylceramide and glucosphingosine as a result of reduced glucocerebrosidase (GCase) enzyme activity due to mutations in the β-glucocerebrosidase gene (GBA).
Conduritol-beta-epoxide (CBE) is a specific inhibitor of GCase activity and can thus be used to induce Gaucher disease in vivo.
C57Bl/6 mice were intraperitoneally injected with 100 mg/kg CBE on 9 consecutive days and brains analyzed for neuroinflammation.
Figure 1. Cortical astrocytosis and activated microglia of CBE-treated mice. GFAP (A) and CD11b (B) immunoreactive (IR) area in percent. n = 9 per group; unpaired t-test; Mean + SEM. ***p<0.001. Representative images of cortical tissue of vehicle-treated or CBE-treated mice. Note the occurrence of extremely enlarged microglia (arrowhead) and increased GFAP in CBE-treated mice.
Schizophrenia
MK-801-Induced Schizophrenia
Non competitive NMDA receptor antagonists, such as MK-801, are shown to produce complex symptoms that mimic positive and negative symptoms, as well as the cognitive deficits of schizophrenia. MK-801 (hydrogen maleate) impairs learning and memory functions that depend on the hippocampus and the amygdala. MK-801 also produces various effects on rodent behavior including deficits in sensory processing, hyperactivity, stereotypy and ataxia. Antipsychotic drugs, such as clozapine, used in the treatment of schizophrenia have been shown to improve MK-801 induced cognitive impairment in mice.
C57Bl/6 mice were injected with 0.2 mg/kg MK-801 and immediately analyzed.
Figure 1: Open field test. A: Distance traversed of MK-801-treated animals compared to sham treated control (CTRL) B: Hyperactivity level of MK-801-treated animals compared to sham treated control. Mean ± SEM; Two-way ANOVA with Bonferroni’s post hoc test. *p<0.05; **p<0.01; ***p<0.001.
Amphetamine (AMPH) or Phencyclidine (PCP) Treated Rat Model
The most widely validated animal models of the positive, negative and cognitive symptoms of schizophrenia involve administration of the dopamine-releasing drug, d-amphetamine in combination with the benzodiazepine Chlordiazepoxide (AMPH) or an open channel NMDA receptor blocker, phencyclidine (PCP). Pretreatment with Clozapine (CZP) can reverse the observed effects.
- AMPH increases activity in the Open Field test 10 minutes after treatment
- AMPH effect can be decreased by CZP
Figure 1: Open field behavior of amphetamine (AMPH)-treated Sprague Dawley rats. A: Distance traversed in cm/5 min over time; Two-way repeated measurements ANOVA with Bonferroni‘s post hoc test. Mean ± SEM. B: distance traversed in cm/5 min, 20 minutes after start of the analyses; One-way ANOVA with Bonferroni‘s post hoc test. Mean + SEM. CTRL = Control; CTRL, AMPH, AMPH+CZP: n = 8-10 per group;. *p<0.05; **p<0.01.
- PCP increases the startle response of Sprague Dawley rats in the prepulse inhibition test, effect can be prevented by CZP
- PCP decreases prepulse inhibition of Sprague Dawley rats in the prepulse inhibition test, effect can be reversed by CZP, effect of PCP depends on prepulse intensity
Figure 2: Startle response and Prepulse inhibition of PCP-treated Sprague Dawley rats. A: Startle amplitude in gram of PCP, PCP+CZP or CZP treated animals at 120 dB; One-way ANOVA with Bonferroni‘s post hoc test. B: Prepulse inhibition in percent of vehicle-, PCP- or PCP+CZP-treated animals using 4 different dB intensities; Two-way ANOVA with Bonferroni‘s post hoc test. A,B: n = 10; Mean + SEM; **p<0.01;***p<0.001.
Neuroinflammation
Lipopolysaccharide (LPS) Induced Neuroinflammation
Neuroinflammation is a common feature of different neurodegenerative diseases, such as Alzheimer’s disease, Parkinson’s disease, Frontotemporal dementia, Amyotrophic Lateral Sclerosis and many more. Recent research suggests that targeting neuroinflammation might present a valid method to treat neurodegenerative diseases. To model neuroinflammation independent from other disease relevant pathologies, mice can be peripherally injected with lipopolysaccharide (LPS). Several different LPS treatment regimes are published. Here we present exemplarily the effect of four 0.5 mg/kg LPS injections on consecutive days.
Other treatment protocols can be performed according to your needs.
Figure 1: Immunofluorescent labeling of the medial hippocampus of LPS-treated mice. Animals were intraperitoneally injected with 0.5 mg/kg LPS (A) or vehicle (B) on 4 consecutive days. Tissue was labeled with CD68 antibody (red) for macrophages, IBA1 antibody (green) for microglia and DAPI (blue) for nuclear staining.
Multiple Sclerosis
Cuprizone-Induced Multiple Sclerosis
Cuprizone is a copper chelator, that causes rapid demyelination and gliosis, and rapid proliferation of glia subtypes. The cuprizone mouse model captures several aspects of Multiple Sclerosis (MS) pathology like demyelination / remyelination, cognitive decline, altered activity and motor deficits.
The cuprizone model is the most frequently used model among the toxin-induced MS models and is used to study mechanisms of oligodendrocyte turnover, gliosis as well as motor capabilities. This animal model is thus suitable to assess certain aspects of the MS pathology and to test pharmaceutical compounds.
C57Bl/6 mice were fed with 0.3 % cuprizone chow for 1 month. Behavioral changes were analyzed within the last week of cuprizone treatment.
Figure 1: Beam walk test, MAO activity and astrocytosis of C57BL/6 mice after 4 weeks of cuprizone treatment. A: latency to traverse a 10 mm wide square beam in seconds. B: MAO activity in brain lysates. C: Quantification of astrocytosis in the hippocampus by GFAP labeling. Mean + SEM; n = 10 per group; unpaired t-test/Mann Whitney test; ***p<0.001.
EAE Mouse Model of Multiple Sclerosis
Experimental autoimmune encephalomyelitis (EAE) shows many pathological similarities to Multiple Sclerosis (MS) and is therefore often used as model to mimic MS by injecting Myelin-Oligodendrocyte-Glycoprotein (MOG) in combination with pertussis toxin (PTX).
The EAE model is widely used as inducible MS model presenting commonly observed MS pathologies like demyelination, neuroinflammation as well as motor strength and coordination.
C57Bl/6 mice were injected with a MOG and PTX regime and clinical signs, motor coordination and spinal cord neuropathology was evaluated.
Figure 1: Clinical signs, motor deficits neurofilament-light chain levels and demyelination of EAE mice. EAE induced C57Bl/6 mice were tested for clinical signs (A), muscle strength in the wire suspension test (B), plasma neurofilament-light chain levels (C) compared to vehicle treated and EAE + Fingo treated mice and Luxol Fast Blue staining for myelin in EAE and Sham induced mice (D). A-C: n = 13 16 per group; Mean + SEM; Kruskal-Wallis One-way ANOVA followed by Dunn‘s multiple comparisons post hoc test; *p<0.05; **p<0.01; ***p<0.001. *EAE-Vehicle vs. Sham-Vehicle; #EAE-Fingo vs. EAE Vehicle. Fingo = Fingolimod.
Amyotrophic Lateral Sclerosis
TDP-43 Induced ALS Mouse Model
TAR DNA binding protein (TARDBP or TDP-43) is shown to play a crucial role in a growing set of neurodegenerative diseases. While strongly related to sporadic and familial forms of Amyotrophic Lateral Sclerosis (ALS), intraneuronal TDP-43 accumulation and aggregation is also related to Frontotemporal Lobar Degeneration (FTLD-TDP or formerly FTLD-U).
To induce ALS in C57BL/6 mice, animals were injected into the motor cortex with adeno-associated viral (AAV) particles of serotype 9 that express human TDP-43 (AAV9-GFP-hTDP-43). Animals were 3 month of age at start and analyzed for 6 months. Control animals were injected with AAV9-GFP. AAV9-GFP-hTDP-43 injected animals show:
- Increased anxiety
- Learning deficits
- Early motor impairments
- Increased neuroinflammation
- Increased ubiquitination
- Neuronal loss
Figure 1: Wire hanging in AAV9-GFP-hTDP-43 and control mice. Latency to fall off the wire was measured. Two-Way-ANOVA with Bonferroni post hoc test. Mean + SEM. Control: n = 16, hTDP-43: n = 20. * p < 0.05, ** p < 0.01, *** p < 0.001.
Figure 2: Western blot analysis of human and total TDP-43 expression in the cortex of AAV9-GFP-hTDP-43 and control mice 6 months after injection. Samples were homogenized in RIPA buffer. GAPDH as loading control. PC: positive control = hippocampal RIPA sample of transgenic TDP-43 mouse. NC: negative control = untreated wild type mouse.
Figure 3: Immunolabeling of GFP (white), human TDP-43 (red), Iba1 (microglia marker, green) and nuclei (DAPI, blue) in the brain of AAV9-GFP-hTDP-43 injected and control mice. Coronally cut brain samples at 0.62 mm. White box: Area of injection magnified at the right. Virus was injected bilaterally into the motor cortex region M1 of 3 months old mice. Mice were sampled 6 months after injection.
QPS Neuropharmacology offers a custom tailored study design for these models and we are flexible to accommodate to your special interests. We are also happy to advice you and propose study designs. Induced models have a explicitly low turn-around time by using wild type mice of commercial breeding companies.
We would be glad to test your compounds in these mouse models!
As with all other in vivo models we are also ready to provide samples (brain tissue, CSF etc.) from these animals for analyses in your laboratory.