TNFA Rat, His Active

What is rat TNF-alpha and what are its primary biological functions?

Rat TNF-alpha is a cytokine that binds to TNFRSF1A/TNFR1 and TNFRSF1B/TNFBR receptors. Structurally, the mature soluble form of rat TNF-alpha typically consists of amino acids 80-235 (according to the P16599 accession) . This protein is predominantly secreted by macrophages and plays multiple crucial roles in both physiological and pathological processes.

The primary biological functions of rat TNF-alpha include:

• Induction of cell death in specific tumor cell lines

• Potent pyrogen activity (fever induction) through direct action or stimulation of interleukin-1 secretion

• Contribution to cachexia development

• Induction of insulin resistance in adipocytes by inhibiting insulin-induced IRS1 tyrosine phosphorylation and glucose uptake

• Promotion of angiogenesis through VEGF production stimulation (synergistically with IL1B and IL6)

• Mediation of bone resorption through osteoclastogenesis promotion

• Induction of IL12 production in dendritic cells via the TNF intracellular domain (ICD)

In experimental settings, TNF-alpha exists in both transmembrane (26 kD) and soluble forms, each potentially exhibiting distinct biological activities, which researchers should consider when designing experiments .

How is the biological activity of recombinant rat TNF-alpha measured?

Biological activity assessment of recombinant rat TNF-alpha typically employs cytotoxicity assays with specific cell lines. Several established methodologies include:

• Cytolytic effect measurement on mouse WEHI-13VAR cells in the presence of Actinomycin D

  • Typical ED50 (effective dose for 50% response): <5 pg/mL

• Cytotoxicity assessment using L-929 mouse fibroblast cells with actinomycin D as a metabolic inhibitor

  • ED50 range: 4-20 pg/mL

• Alternative bioassays utilizing murine cell lines like WEHI 164 clone 13

For accurate activity determination, researchers should perform a dose-response assay to identify optimal concentrations for their specific experimental conditions . It's important to note that while bioassays detect only biologically active TNF-alpha, ELISA methods can detect both active and inactive forms, which may be significant for comprehensive studies examining regulatory mechanisms .

What expression systems are used for producing recombinant rat TNF-alpha proteins?

Multiple expression systems are employed to produce recombinant rat TNF-alpha, each offering distinct advantages:

The selection of an appropriate expression system should be guided by specific experimental requirements, as the biological properties of the recombinant protein—including glycosylation patterns, folding characteristics, and activity levels—can vary significantly between systems .

What is the difference between membrane-bound and soluble forms of rat TNF-alpha?

Rat TNF-alpha exists in two primary forms with distinct structural and functional characteristics:

• Membrane-bound form:

  • Approximately 26 kD transmembrane protein

  • Expression is activity-dependent in neurons

  • When facial whiskers are repeatedly stimulated, neuronal production of transmembrane TNF increases in the somatosensory cortex

  • May participate in reverse signaling (where cells expressing membrane TNF receive signals)

• Soluble form:

  • Typically encompasses amino acids 80-235 of the full protein

  • Molecular weight around 17-19 kD after processing

  • Primarily secreted by macrophages

  • Forms biologically active trimeric complexes with TNF receptors

  • More readily measurable in plasma using cytotoxic assays

The conversion from membrane-bound to soluble form occurs through proteolytic cleavage. Both forms possess biological activities, but their signaling capabilities and downstream effects can differ significantly. The membrane-bound form primarily facilitates juxtacrine signaling (cell-to-cell contact), while the soluble form enables paracrine or endocrine signaling at greater distances .

How should recombinant rat TNF-alpha be reconstituted and stored for optimal activity maintenance?

Based on established protocols, the following recommendations ensure optimal activity maintenance of recombinant rat TNF-alpha:

Reconstitution guidelines:

• Reconstitute lyophilized material in 0.5 mL of distilled or deionized water

• For further dilution, use buffers containing carrier protein:

  • PBS with 1% BSA, or

  • Neutral media containing <10% FCS

• Alternative reconstitution buffer: 20mM Tris, 150mM NaCl (pH 8.0) to a concentration of 0.1-1.0 mg/mL (avoid vortexing)

• Some products are supplied in specific formulations:

  • 20mM Tris, 150mM NaCl, pH 8.0, containing 1mM EDTA, 1mM DTT, 0.01% SKL, 5% Trehalose and Proclin300

Storage recommendations:

• Store lyophilized material at 4°C

• After reconstitution, aliquot to minimize freeze-thaw cycles

• Store aliquots at or below -70°C/-80°C for long-term stability

• Short-term storage at 2-8°C is possible for up to one month

Quality control metrics indicate that properly stored recombinant rat TNF-alpha should show less than 5% activity loss within the expiration date, as determined by accelerated thermal degradation tests (37°C for 48h) showing no obvious degradation or precipitation .

How does TNF-alpha administration affect neuroinflammation in different rat strains?

The neuroinflammatory response to TNF-alpha administration varies significantly between rat strains, particularly between Dahl salt-sensitive (Dahl-S) rats and Sprague-Dawley (SD) rats:

Baseline differences:
Dahl-S rat neurons express higher constitutive levels of inflammatory markers compared to SD rats, including:

• CCL5

• IL-1β

• iNOS

• NF-κB subunit nfkb1

Differential responses to TNF-alpha:
When exposed to TNF-alpha, cultured neurons from Dahl-S rats demonstrate significantly augmented inflammatory responses compared to SD rat neurons, with greater upregulation of:

• CCL5

• IL-1β

• IL-16

• iNOS

In vivo, central administration of TNF-alpha (250 ng via ICV injection) increases immunoreactivity of inflammatory markers in the paraventricular nucleus (PVN) of both rat strains, but with strain-specific patterns. Dahl-S rats show particularly elevated responses in CCL12 and IL-1β compared to SD rats .

These findings suggest that Dahl-S rats possess an inherently heightened sensitivity to TNF-alpha-induced neuroinflammation, likely due to their elevated baseline NF-κB expression. This enhanced inflammatory phenotype, combined with the impaired blood-brain barrier function documented in Dahl-S rats, may contribute to their increased vulnerability to neuroinflammatory conditions and hypertension development when exposed to inflammatory stimuli .

What are the optimal protocols for studying TNF-alpha effects on neuronal activity in rat models?

For investigating TNF-alpha effects on neuronal activity in rat models, several methodological approaches have proven effective:

Dosage considerations:

• For intracerebroventricular (ICV) injection: 250 ng TNF-alpha dissolved in 2.5 µl saline

• For intravenous or intra-carotid administration: 0.5 μg/kg

• These doses are based on established protocols showing significant physiological responses without excessive toxicity

Administration routes:

• Central administration (direct neural effects):

  • ICV injection for broad central nervous system exposure

  • Direct microinjection into specific brain nuclei (e.g., PVN) for localized effects

• Peripheral administration (blood-borne effects):

  • Intravenous injection for systemic circulation

  • Ipsilateral carotid artery (ICA) injection for preferential delivery to brain vasculature

Experimental timeline:

• For acute neuroinflammatory responses: Collect tissue 3 hours post-TNF-alpha administration

• For neuronal activation studies: Record parameters (neuronal firing, sympathetic nerve activity) continuously from administration through peak response (typically 15-60 minutes)

Critical controls:

• Vehicle administration (e.g., saline) matching the volume and route of TNF-alpha delivery

• Pathway-specific interventions:

  • Vagotomy to eliminate vagal afferent contributions

  • Mid-collicular decerebration to isolate brainstem mechanisms

  • Cyclooxygenase inhibitors (e.g., ketorolac) to assess prostaglandin-mediated effects

These protocols enable comprehensive investigation of both direct and indirect mechanisms by which TNF-alpha influences neuronal activity across different brain regions, with particular relevance to autonomic and neuroendocrine regulation.

How can researchers distinguish between active and inactive forms of TNF-alpha in rat experimental models?

Differentiating active from inactive TNF-alpha in rat experimental models requires combining complementary methodological approaches:

Bioassay approach:

• Employs cytotoxic assays using murine cell lines (L929 or WEHI 164 clone 13)

• Specifically detects only biologically active TNF-alpha

• Typical sensitivity: ED50 of 4-20 pg/mL

• Advantages: Directly measures functional activity

• Limitations: Misses inactive forms that may have regulatory significance

ELISA-based detection:

• Utilizes antibodies that recognize epitopes present in both active and inactive forms

• Detects total TNF-alpha (active + inactive)

• Lower detection limit: approximately 39.0 pg/mL

• Inter- and intra-assay coefficients of variation typically <12.0%

• Specificity confirmed by parallelism between recombinant murine TNF and rat TNF standards

Combined analytical approach:
For comprehensive assessment, researchers should employ both methods in parallel:

• Measure total TNF-alpha concentration via ELISA

• Determine active TNF-alpha fraction through bioassay

• Calculate inactive TNF-alpha proportion: Total TNF-alpha (ELISA) - Active TNF-alpha (bioassay)

This integrated approach provides valuable insights into TNF-alpha regulation, particularly in models of acute infection and inflammation where the balance between active and inactive forms may have significant physiological implications .

What mechanisms mediate the cardiovascular effects of blood-borne TNF-alpha in rat models?

Blood-borne TNF-alpha exerts cardiovascular effects through specific neural and biochemical pathways:

Key brain regions involved:

• Paraventricular nucleus (PVN) of hypothalamus

• Rostral ventrolateral medulla (RVLM)

• These regions are critical for sympathetic regulation and cardiovascular control

Activation pathway:

• TNF-alpha in circulation (0.5 μg/kg administered intravenously or via ipsilateral carotid artery)

• Central prostaglandin synthesis (particularly PGE2)

• Activation of PVN and RVLM neurons

• Increased sympathetic nerve activity

• Resultant cardiovascular responses:

  • Elevated arterial pressure

  • Increased heart rate

Mechanistic evidence:

• Vagotomy does not affect responses to intravenous TNF-alpha, indicating non-vagal pathways

• Mid-collicular decerebration reduces responses, suggesting involvement of supramedullary structures

• Injection of cyclooxygenase inhibitor ketorolac (150 μg) into lateral ventricle substantially reduces cardiovascular responses to TNF-alpha

• Direct PGE2 (50 ng) administration into lateral ventricle or PVN produces similar but faster-onset responses compared to TNF-alpha

This mechanistic pathway explains how peripheral inflammatory signals communicate with specific brain regions to influence autonomic function and cardiovascular regulation, with central prostaglandins serving as crucial mediators between the peripheral inflammatory signal (TNF-alpha) and central autonomic responses .

How does TNF-alpha influence sleep regulation in rat models, and what methodologies best capture these effects?

TNF-alpha plays a significant role in sleep regulation, as evidenced by both direct research on TNF-alpha and studies using TNF-alpha-blocking medications:

TNF-alpha's role in sleep biology:

• Expression in the brain is activity-dependent

• Neuronal production of transmembrane TNF-alpha (26 kD) increases in the somatosensory cortex following sensory stimulation

• TNF-alpha levels fluctuate with sleep-wake cycles

Effects of TNF-alpha modulation on sleep:

• TNF-alpha receptor antagonism with Etanercept (ETA) improves multiple sleep parameters:

  • Enhanced sleep efficiency

  • Increased total sleep time

  • Extended stage 2 sleep duration

  • Reduced daytime sleepiness

Recommended methodological approaches for rat studies:

• Sleep architecture analysis:

  • Polysomnography with EEG/EMG recording in freely moving rats

  • Quantification of NREM sleep, REM sleep, and wakefulness

  • Analysis of sleep fragmentation and transition patterns

• Administration protocols:

  • Central (ICV) administration to study direct CNS effects

  • Peripheral administration to model systemic inflammation effects

  • Dose-response studies to determine threshold and ceiling effects

• Molecular correlates:

  • Brain region-specific TNF-alpha expression across sleep-wake cycles

  • Receptor expression and signaling pathway activation

  • Interaction with other sleep-regulatory molecules (e.g., IL-1β, adenosine)

• Interventional studies:

  • TNF-alpha receptor blockade effects on sleep in both normal and pathological conditions

  • Comparative effects of selective TNFR1 vs. TNFR2 antagonism

  • Sleep recovery patterns following sleep deprivation with TNF-alpha modulation

These approaches would help elucidate the complex mechanisms through which TNF-alpha influences sleep architecture and regulation, potentially informing therapeutic strategies for sleep disorders associated with inflammatory conditions.

What considerations are important when using recombinant rat TNF-alpha for blood-brain barrier studies?

When investigating TNF-alpha effects on the blood-brain barrier (BBB), several important considerations should guide experimental design:

Strain-specific BBB characteristics:

• Dahl salt-sensitive (Dahl-S) rats show impaired BBB function compared to Sprague-Dawley (SD) rats

• This differential barrier integrity affects how TNF-alpha and other inflammatory molecules access the brain

• These strain differences may influence experimental outcomes and interpretation

Administration routes for BBB interaction studies:

• Systemic administration (intravenous):

  • 0.5 μg/kg TNF-alpha for studying how circulating cytokines affect brain through intact BBB

  • Allows assessment of transport mechanisms and barrier penetration

• Carotid artery injection:

  • More direct delivery to cerebral vasculature

  • Reduces peripheral effects while maximizing BBB exposure

• Dual-compartment administration:

  • Combined peripheral and central administration at different doses/timepoints

  • Helps distinguish direct vs. BBB-mediated effects

Mechanistic pathways across the BBB:

• Blood-borne TNF-alpha activates paraventricular nucleus (PVN) and rostral ventrolateral medulla (RVLM) neurons

• This activation involves prostaglandin-mediated pathways

• The temporal dynamics of direct PGE2 injection versus blood-borne TNF-alpha effects provide insights into BBB transport kinetics and secondary messenger systems

Analytical approaches:

• Immunohistochemistry for tight junction proteins and transporters

• Quantitative assessment of BBB permeability using labeled tracers

• Measurement of inflammatory marker expression on both luminal and abluminal sides of the BBB

• Correlation between TNF-alpha levels and BBB functional parameters

These considerations facilitate rigorous investigation of how TNF-alpha modulates BBB function, with implications for understanding neuroinflammatory mechanisms in various pathological conditions.

How can researchers optimize rat TNF-alpha activity assays for consistent results across different experimental designs?

Optimizing rat TNF-alpha activity assays requires attention to several critical factors:

Standardized cell line selection:

• WEHI-13VAR or L-929 mouse fibroblast cells are commonly used

• Maintain consistent passage numbers (typically between passages 5-20)

• Regular mycoplasma testing to ensure cell line integrity

• Standardized cell density (typically 2-4 × 10^4 cells/well)

Assay conditions optimization:

• Actinomycin D concentration:

  • Typically 1 μg/mL, but should be optimized for each cell batch

  • Pre-incubation period: 3-4 hours before TNF-alpha addition

• Incubation parameters:

  • Temperature: 37°C

  • CO2 concentration: 5%

  • Humidity: >95%

  • Duration: 18-24 hours (optimize for maximum sensitivity)

Standard curve development:

• Use recombinant rat TNF-alpha from a consistent source

• Prepare fresh standards for each assay run

• Generate standard curves covering 1-1000 pg/mL

• Validate parallelism between standards and biological samples

• Include quality control samples with known activity levels

Result normalization strategies:

• Express results as percentage of control cell death

• Determine ED50 (effective dose producing 50% maximum response)

• Convert to activity units: 1 unit typically defined as ED50 in pg/mL

• For complex biological samples, include reference standards for inter-assay comparison

Validation protocols:

• Inter-assay coefficient of variation goal: <15%

• Intra-assay coefficient of variation goal: <10%

• Spike recovery tests: 80-120% recovery

• Parallelism between dilution curves of standards and samples

Following these optimization strategies enables researchers to achieve consistent and reproducible TNF-alpha activity measurements across diverse experimental conditions, facilitating meaningful comparisons between studies.

What methodological approaches are recommended for investigating TNF-alpha-induced changes in sympathetic nerve activity?

For investigating TNF-alpha effects on sympathetic nerve activity, the following methodological approaches are recommended:

Animal preparation:

• Urethane anesthesia (1.2-1.4 g/kg, ip) provides stable long-term recording conditions

• Tracheotomy for airway management and mechanical ventilation if necessary

• Femoral artery cannulation for continuous blood pressure monitoring

• Temperature maintenance at 37°C using heating pad

TNF-alpha administration protocols:

• Intravenous administration:

  • Dose: 0.5 μg/kg TNF-alpha

  • Delivery: Slow infusion over 1-2 minutes

  • Vehicle control: Equivalent volume of saline

• Ipsilateral carotid artery injection:

  • Same dose (0.5 μg/kg)

  • Allows more directed delivery to brain vasculature

Neural pathway investigation techniques:

• Vagotomy: Bilateral cervical vagus nerve transection to eliminate vagal afferent signaling

• Mid-collicular decerebration: Surgical separation of forebrain from brainstem to isolate brainstem-mediated effects

• Pharmacological interventions:

  • Lateral ventricle injection of cyclooxygenase inhibitor ketorolac (150 μg)

  • Direct PGE2 (50 ng) administration into lateral ventricle or PVN

Physiological recording parameters:

• Sympathetic nerve activity:

  • Target nerve: Usually renal sympathetic nerve

  • Recording technique: Multifiber recording using bipolar silver wire electrodes

  • Signal processing: Amplification (10,000-20,000×), filtering (30-3000 Hz), full-wave rectification, and integration

• Cardiovascular parameters:

  • Continuous arterial pressure monitoring

  • Heart rate determination from pulse pressure

  • Calculated parameters: Mean arterial pressure, pulse pressure

• Neuronal activation assessment:

  • Single-unit or multiunit recording from PVN and RVLM neurons

  • Classification of neurons based on firing patterns and responses to physiological challenges

This comprehensive methodological approach enables systematic investigation of the pathways and mechanisms through which TNF-alpha modulates sympathetic nerve activity and cardiovascular function, with implications for understanding inflammatory mechanisms in autonomic dysregulation.