Table 2:

Common In vivo Models of Neutrophil Function.

Assay	Type, Application or Benefit	How to	Difficulties	Reference
Cremaster (IVM)	- Most common for studying recruitment cascade - Only transillumination needed, possibly fluorescence imaging and RLOT - Administration of inflammatory stimuli possible (trauma vs. TNFα vs. fMLP vs. LPS) → local inflammation - CLP for systemic inflammation	- Inject stimulus (e.g. TNFα intrascrotally) - Wait 2h - Anesthetize mouse - Mobilize cremaster	- Tissue damage and flow reduction by stretching of the muscle - Bleeding	- [128]
Lung (IVM)	Intravital microscopy	- Anesthetize mouse, mechanical ventilation - Thoracic window preparation	- Motion artefacts - Bleeding - Tissue damage due to thoracic window	- [129]
Lung inflammation	- Different disease models ((A) ventilator induced VILI vs. (B) acid induced ALI vs. (C) pneumonia)	- (A) Mechanical ventilation (VILI) - (B) Application of acid (ALI) - (C) Application of bacteria (intratracheal injection) - (C) Bacterial burden (plating of tissue samples) - (A–C) Flow cytometry of whole organ lysates and bronchoalveolar lavage for inflammatory cell recruitment	- Inhomogeneous injection of bacteria (left vs. right lung)	- (A) ALI. [130] - (B) Bacterial pneumonia:. [11] - (C) LPS induced lung injury: [69]
Heart (IVM)	Intravital microscopy	- Anesthetize animal - Intubation and mechanical ventilation - Preparation of thorax - Connect ECG - Positioning of suction device or imaging-holder - ECG-triggered imaging	- Motion artefacts - Bleeding - Tissue damage	- Heterotopic heart transplant: [43] - Tissue stabilization and acquisition gating: [131]
Heart inflammation	- LAD occlusion - Lipotoxic cardiomyopathy	- Anesthesia - Thorax preparation - Ligation of the LAD - Re-opening of LAD if wanted	- Inconsistent vessel ligation	- LAD occlusion: [93] - Myocarditis: [132] - Lipotoxic cardiomyopathy: [133] - Sepsis related cardiac dysfunction: [134] - Model guidelines: [135]
Kidney (IVM)	Intravital microscopy	- Anesthesia - Preparation of kidney - Intravital microscopy	- Bleeding - Non-flat surface for imaging	- [136]
Kidney inflammation	(A) Ischemia reperfusion injury vs. (B) Sepsis related acute kidney injury vs. (C) Chronic kidney failure	(A) - Clamping of both kidneys (B) - Uterine ligation and bacterial inoculation or - CLP (C) - 5/6 nephrectomy	(A) - Bleeding, organ damage (B) - Inconsistent cecal puncturing or bacterial burden (C) - Inconsistent nephrectomy size	- (A) IRI: [44] - (B) Sepsis related: Uterine ligation and bacterial inoculation:. [137] - (B) Sepsis related: CLP: [138] - (C) CKD: [124]
Brain (IVM)	Intravital microscopy, cranial window	- Anesthesia - Disinfect and prepare skin - Thinning of skull bone with micro-drill - Multiphoton imaging	- Tissue damage, compression artefacts - Bleeding	- [139]
Brain inflammation	(A) CLP vs. (B) EAE vs. (C) Stroke	(A) Prepare abdomen, ligate and puncture the cecum (B) Subcutaneous injection of Mog35–55 peptide in CFA with 1 mg/ml killed Mycobacterium tuberculosis H37Ra; double injection of i.v. pertussis toxin. (C) MCA electrocoagulation or small filament insertion	(A) Inconsistent ligation and puncture (B) Scoring difficulties, incongruent resemblance of human MS disease (C) Inconsistent advancement of filament or tissue damage by electrocoagulation	- (A) [140][91] - (B) [141] - (C) MCA electrocoagulation: [89]; Filament insertion: [142]
Liver (IVM)	Intravital microscopy	- Anesthesia - Opening of abdominal cavity - Mobilization of liver lobe - Tissue preparation and coverslip attachment - Time lapse microscopy	- Immobilization - Drying of liver tissue - Duration of imaging	- [143]
Liver inflammation	(A) Bacterial vs. (B) Sterile heat injury	(A) CLP (compare above) or injection of bacteria (B) - Anesthesia - Opening of abdominal cavity - Mobilization of liver lobe - Heat wire injury - Time lapse microscopy	(A) Compare above (B) Inconsistent injury size; excessive tissue damage	-(A) MRSA infection: [76] -(B) [41]
Intestinal inflammation	Colitis (A) Bacterial (B) Dextran sodium sulfate (DSS)-induced	(A) Antibiotic treatment, followed by gavage of bacteria (B) 1.5 to 3.5% DSS in drinking water for 5 to 9 days	- Motility - Inconsistent inflammation	- (A) [144] -(B) [145, 146]
Fetal (Yolk-sac) (IVM)	Yolk-sac vs. skin vs. skull	- Anesthesia - Prepare abdomen - Exteriorize and incise uterus - Mobilize fetus - Imaging	- Drying - Tilted surface - Pressure onto vessels	- [147]
Joints	Arthritis model	- Serum separation from K/BxN mice - Injection of serum (150–250 μl) intraperitoneally on days 0 and 2 - Clinical scoring	- Inconsistent injections - Inconsistent blood collection and serum separation from donor mice	- [148][149]
Skin (IVM)	Intravital microscopy	- Anesthesia - Prepare imaging area, for example ear stage - Multiphoton microscopy	- Movement artefacts - Pigmentation - Z-deviation	- [150]
Skin inflammation	(A) Bacterial vs. (B) Allergic skin inflammation	(A) Intradermal injection in small volume (0.5 – 4 μl) into pinnae of ear or back skin of mice (B) Shave skin, tape-strip 6 times, place patch with chicken egg ovalbumin on skin; sensitization for 7 weeks, challenge on days 0 and 3 after sensitization by application of ovalbumin to shaved, tape-stripped, nonharmed skin area	- Inconsistent stimulation - Injection of amount of bacteria imprecise or displacement of ovalbumin patch	-(A) [151] -(B) [152]
Atherosclerosis	ApoE knockout model	- ApoE−/− mice - High fat diet containing 21% fat (ssniff)	- Dissimilar lipid metabolism to humans (VLDL vs. LDL) - Possible non-atherosclerosis related inflammatory effects	- [153]
Venous thrombosis	Flow restriction model	- Anesthesia - Median laparotomy - Placement of space holder on inferior vena cava - Application of narrowing ligature below left renal vein - Removal of space holder - Flow measurements (reduction by approx. 80% desired)	- Injury of the vessel wall and subsequent thrombus induction due to endothelial damage - Bleeding - Complete occlusion of the vessel	- [126]
Systemic hypoxia	Normobaric hypoxia supply	- Expose animals to hypoxic conditions for set time periods (e.g. 4 hours) - Can be combined with other disease models (such as bacterial pneumonia)	- Inconsistent oxygen supply - Changes in respiratory rate - Differences in gas-mixtures used for hypoxia induction	- [50][48]

Abbreviations Table 2: CFA: Complete Freund’s adjuvant; CLP: Cecal ligation puncture; DSS: Dextran sulfate sodium; EAE: Experimental autoimmune encephalomyelitis; LAD: Left anterior descending; MCA: Middle cerebral artery; MRSA: Methicillin resistant staphylococcus aureus; MS: Multiple sclerosis; RLOT: Reflected light oblique transillumination; VILI: Ventilator induced lung injury, ALI: acute lung injury.