Shanklin, D. Radford
Mullins, Amanda C.
Baldwin, Heather S.
Ventilatory treatment of neonatal respiratory distress often results in bronchopulmonary dysplasia
from congenital surfactant deficiency due to mutants of transporter protein ABCA3. Association
of this condition with other severe disorders in premature newborns has not heretofore been
reported. A neonatal autopsy included an in vivo whole blood sample for genetic tcsting.
Autopsy revealed severe interstitial pulmonary fibrosis at age 8 days with heterozygotic mutation
p.E292V of ABCA3 and severe dystrophic retardation of cerebral cortcx and cerebellum.
Subsequently, 1300 archival neonatal autopsies, 1983-2006, were reviewed for comparable
concurrent findings and bronchopulmonary dysplasia or retarded cerebral dystrophy lacking the
other principal feature of this syndrome. Archival review revealed four similar cases and eight
less so, without gene analysis. Further review for bronchopulmonary dysplasia revealed 59 cases,
1983-2006. Several other examples of similar retarded migration of germinal matrix and
underdevelopment of cortical mantle, without pulmonary lesions of this type, were identified. The
determination of an ABCA3 mutation in one case of severe pulmonary fibrosis with significant
dystrophy of the brain and the identification of four highly similar archival cases and eight others
with partial pathological findings supports the designation of an independent disorder, here
referred to as the cerebroprrlmonary dysgenetic syndrome.
Shanklin, D. Radford
Normally developed thyroid function is critical to the transition from fetal to neonatal life with the
onset of independent thermoregulation, the most conspicuous of the many ways in which thyroid
secretions act throughout the body. A role for thyroid secretions in growth and maturation of the
lungs as part of the preparation for the onset of breathing has been recognized for some time but how
this contributes to tissue and cell processes and defenses under the duress of respiratory distress has
not been well examined. Extensive archival autopsy material was searched for thyroid and adrenal
weights, first by gestational age, and then for changes during the first hours after birth as ratios to
body weight. After a gestational age of 22 weeks the fetal thyroid and adrenal glands at autopsy in
those with hyaline membrane disease are persistently half the size of those in "normal" infants dying
with other disorders. When the thyroid is examined shortly after birth it reveals a post natal loss of
mass per body weight of similar orders of magnitude which does not occur in the control group. A
clinical sample of premature infants with (12) and without (14) hyaline membrane disease was
tested for T4, TSH, TBG, and total serum protein. The results also demonstrate a special subset with
lower birth weights at the same gestational age, and lower serum T4 and total serum protein. Ventilatory distress in newborn rabbits was induced by bilateral cervical vagotomy at 24 hours post
natal following earlier injection of thyroxine (T4) or thyroid stimulating hormone (TSH) and
comparisons were made with untreated animals and by dose. Early life thyroidectomy was
performed followed by exposure to either air or 100% oxygen. A final experiment in air was
vagotomy after thyroidectomy. Composite analysis of these methods indicates that thyroid factors
are both operative and important in the newborn animal with ventilatory distress. This work and
the archival data indicate those infants destined to develop hyaline membrane disease through
respiratory distress are a distinct developmental and clinical subset with the point of departure from
otherwise normal development and maturation in the second or early third trimester. This interval
is known to be a period of marked variation in the overview indicators of fetal progress through
gestational time. The initiating factor or circumstance which then separates this special subset from
normal future development is placed by these observations firmly into the period when human fetal
TSH dramatically rises 7-fold (17.5-25.5 weeks) followed by a lesser 3 to 4 fold increase in T4
which is extended into the early third trimester. The earlier part of this interval is characterized by
the thyrotrophic action of chorionic gonadotropin (hCG). The possibility that abnormalities in the
intrauterine environment secondary to maternal infection play a role within this time frame is
indicated by the demonstration that interleukin-2 (IL-2) induces an anterior pituitary release of
TSH. Since IL-2 has this property and is not an acute phase cytokine, some form of chronic
infection or an immunopathic process seems more likely as a possible active factor in pathogenesis.
Shanklin, D. Radford
Oxygen uptake by the pulmonary circulation is a chemical reaction. The physicochemical attributesof oxygen are critical when studying pulmonary oxygen toxicity. Extent of lung injury depends onthe percentage of oxygen in an oxygen:nitrogen mix in polybaric circumstances (Shanklin, 1969). Further change in extent of lesion follows when other gases are used in the inhalant mix instead of nitrogen (Shanklin and Lester, 1972), with oxygen at 21-100% of the mix. Comparative subatmospheric oxygen levels down to 3% in hydrogen, helium, nitrogen, argon, or sulfurhexafluoride, were run with and without ventilatory distress by the Farber (1937) model, bilateralcervical vagotomy (BCV). This yielded coherent results indicating a need to consider molecular characteristics at the atomic level. Molecular mass and size, gas viscosity, and thermal conductivity yielded no obvious correlates to lung injury. Saturation of the outer electron shells of the diluents fit the empiric data, prospectively an interaction between oxygen and nitrogen from their electronegativity and closely approximate molecular mass, size, and shape. The lesion is essentially eliminated at 7% oxygen in nitrogen. At 3% oxygen, the least lesion is found with N2, H2, and SF6,all gases with incomplete outer electron shells, allowing for transient, possibly polarized, covalent bonding with oxygen as the significant minority component in the mix. Argon and helium do not interfere with oxygen. With 3% oxygen in argon without BCV, the experiments ran so long (>70hours) they were terminated once the point had been made. 3% oxygen in argon after BCV yielded a mean survival more than twice that of BCV in air, indicating a remarkable degree of nitrogen interference with oxygen in the respiratory medium of terrestrial animal life. Argon displayed other advantages for the lung compared to nitrogen. Hydrogen, nitrogen, and oxygen are diatomic molecules, a feature which does relate to the extent of lung injury, but only oxygen is paramagnetic. Magnetic effects on lesion formation were tested:  with ventilatory distress induced in newbornrabbits, and  in young adult female white mice exposed to 100% oxygen without addedmechanical distress. A noninvasive model for ventilatory distress, thoracic restraint (TR), withlonger mean survivals of 40-50 hours, was employed rather than the Farber model. Parallel runs
with TR, one subset receiving 100% oxygen in a plastic chamber resting on six strong ring magnetswith measured fields up to +1200 gauss, the other plain 100% oxygen, were performed. Bothsubsets developed moderate metabolic acidosis with average weight losses circa 25%, but over different time courses, 82.89 ± 4.91 hours in magnetized oxygen, 55.4 per cent longer than the 53.34 ± 9.82 hours in plain oxygen ( p <0.001). The longer survival in magnetized oxygen meant extensive lung injury (99.57 ± 0.42% pleural surface, versus 83.86 ± 14.03%), but the rate of lesionformation was 30.89 per cent faster in plain oxygen (1.5722% per hour) than in magnetized oxygen(1.2012% per hour), a difference significant at p <0.001. The effect of oxygen without mechanical ventilatory distress was examined in female adult whitemice exposed to oxygen or magnetized oxygen. Similar survivals and weight losses were achieved. The rate of lung lesion formation was different, 1.2617% per hour in plain oxygen, 46.13 per centfaster than 0.8634% per hour in magnetized oxygen. A variable magnetic field, with animals moving and breathing in chambers flooded with oxygen, has both systemic and pulmonary effectswhich alter the rate of lesion formation due to oxygen toxicity. Paramagnetic oxygen in a magneticfield influences the effect of oxygen toxicity on the lung but at these strengths of field it does notovercome significant mechanical disturbance.
Shanklin, D. Radford
Oxygen is central to the development of neonatal lung injury. The increase in oxygen exposure of
the neonatal lung during the onset of extrauterine air breathing is an order of magnitude, from a
range of 10-12 to 110-120 Torr. The contributions of oxygen and the volume and pressure
relationships of ventilatory support to lung injury are not easily distinguished in the clinical setting.
Sequential changes in inspired air or 100% oxygen were studied in 536 newborn rabbits without
ventilatory support. Bilateral cervical vagotomies (BCV) were performed at 24 hours post natal to
induce ventilatory distress which eventuates in hyaline membrane disease. The sequences applied
yielded evidence for an induced state of oxygen dependency from oxygen use which was reflected
in differences in survival and the extent of pulmonary injury. The median survival for animals kept
in air throughout was 3 hours. Oxygen before vagotomy or during the first 3 hours afterwards
extended the survival significantly but produced more extensive, more severe, and more rapid lung
lesions. Returning animals to air after prior oxygen exposure reduced the number of survivors past
10 hours and shortened the maximum survival in those groups. These features indicate the
development of a dependency of the defense mechanisms on the availability of oxygen at the higher
level for metabolic and possibly other aspects of the pulmonary and systemic response to injury,
beyond the usual physiological need. Subset analysis revealed additive and latent effects of
oxygen and demonstrated a remarkable rapidity in onset of severe lesions under some
circumstances, illustrating the toxicity of oxygen per se.
Shanklin, D. Radford
Dizygotic twins at 38 weeks with separate placentas: twin A, a 2479 gram female, was healthy after vaginal delivery. Five minutes later when the amnion of twin B was ruptured artificially, the cord prolapsed and could not be repositioned. Some 25 minutes later a 2791 gram male was delivered by section. Brain injury was noted soon afterward and subsequent development was marked by severe cerebral palsy and mental retardation. Initial diagnosis of twin B's placenta was 'chorangiosis,' overlooking fresh thrombi blocking the umbilical vein and one umbilical artery. Subsequent assessment revealed the same change in twin A's placenta. Archival records had 18/500 (3.6%) stillborns and 17/418 (4.07%) newborns with central placental hypervascularity. Of 125 recent consult placentas there were 17/100 singleton and 11/25 (44%) twin placentas displaying this change. Of 229 section deliveries there were 0/42 stillborns and 5/187 newborns with this vascular pattern. Another set of 625 autopsies revealed none with both hypoxic encephalopathy and this placental finding. This structural change is the same often seen in placentas from high altitude such as in Denver. Cerebral palsy occurs less often in Colorado than in other American states, per epidemiological data.
Shanklin, D. Radford
Molecular interaction can be determined from biological experiments [MARM 2011, #415, p. 252]. Atomic attributes can be shown to be determinative in whole animal experiments under appropriatecircumstances [MARM 2012, #225, p. 169]. The dynamics of replenishing gas interchange in thedistal air spaces of the mammalian lung, and at the atmosphere-lung interface, are shown bydifferences in the extent of pulmonary lesions after the induction of respiratory distress and changesin the mix of the gases inhaled [Biol.Neonat. 20:140-158, 1972]. Such effects have significantbiological and pathogenetic consequences. Hyaline membrane disease (HMD) is a common andsometimes lethal disorder, especially in premature newborns [Clin.Med. 72:477-490, 1965;Int.J.Clin.Pharmacol. 5:20-25, 1971].
There is significant evidence the lesions can be induced by oxygen enrichment [New Eng.J.Med.277:833-837, 1967; Lab.Invest. 21:439-448, 1969]. Bilateral cervical vagotomy (BCV) is a standardmethod of inducing ventilatory distress which leads to HMD [J.Exp.Med. 66:397-404, 1937;Biol.Neonat. 6:340-360, 1964; Biol.Neonat. 11:61-86, 1967]. This model has relatively short median(2.50-7.22 hours) and mean (3.54-13.63 hours) post-BCV life spans [Lab.Invest. 21:439-448, 1969],making it difficult to identify subtle but important effects which might change the result. Thus, aslower model inducing ventilatory distress, previously studied, was again employed, thoracicrestraint (TR). In this model, quarter inch soft cloth adhesive tape is tightened around the lower ribcage of newborn rabbits, reducing the segmental thoracic circumference by 10%, and then placingthem  in a 480 ml clear plastic chamber with 100% oxygen running at 1.0L/min, or  in anidentical chamber resting on four adherent donut magnets with a varied field up to +1200 gauss. Parallel experiments were done using young adult female white mice to eliminate the effect ofventilatory distress induced by TR.
Diatomic oxygen is the only gas in the inhalant mixtures noted which is inherently paramagnetic.
Studies have considered the effects of magnetic fields on flame combustion which is a chemicalreaction involving oxygen [IEEE Trans.Mag. 21:2077-2079, 1985; IEEE Trans.Mag. 23:2752-2754,1987; J.Appl.Phys. 69:2734-2736, 1991; Combus.Flame 93:207-214, 1993]; oxygenation incapillaries [Int.J.Math. Anal. 4:1697-1706, 2010]; and also on organic photochemical reactions[Acc.Chem.Res. 13:369-377, 1980], which, taken together, indicate magnetic influences on the flowand orientation of oxygen as gas and in solution. Two principal objective results from theseexperiments demonstrate an effect of the magnetic field on the whole animal and on the extent oflung injury. The newborn rabbit survival in pure 100% oxygen was 58.56 ± 3.19 hours versus 82.89± 4.91 hours in magnetized oxygen ( p <0.0001); the difference in gross lung injury was 47.46 ± 6.51per cent versus 99.57 ± 0.43 per cent respectively (p<0.0001). The adult female mice in pure 100%mean survival was 53.71 ± 5.40 hours versus 64.57 ± 2.93 hours in magnetized oxygen (p≈0.015);the respective percentages of lung injury were 61.67 ± 10.91 and 55.75 ± 10.45 (n.s.). But, when theresult is considered on the basis of rate of lung injury per hour, magnetized oxygen is much slower. The rates for newborn rabbits were, (magnetized oxygen) 1.3728%/hour and (plain oxygen),1.7969%/hour, a ratio of 1.3088, or 24.6% slower. In mice the rates were 0.8634%/hour and1.2617%/hour respectively, a ratio of 1.462, or 31.5% slower.
A variable magnetic field added to whole animal models of pulmonary oxygen toxicity changes theoutcome in two overt ways:  survival is enhanced, but despite this,  the rate of formation oflung injury is reduced by 24% in newborn rabbits and by 31% in young adult female white mice. Thus, the toxic effect of oxygen is reduced systemically and in the lung by low strength magneticfield effects on inhaled paramagnetic 100% oxygen.
Shanklin, D. Radford
Molecular interaction can be determined from biological experiments. In the case of dynamics at
the atmosphere-lung interface the physicochemical and atomic attributes of inhalant gases has
significant biological and pathogenetic consequences. Hyaline membrane disease (HMD) is a
common and sometimes lethal disorder, especially in premature newborns. Current therapy includes
artificial ventilation and increased oxygen in the inspired air, despite evidence the lesions can be
induced by oxygen enrichment [Lab.Invest. 21 439, 19691. Bilateral cervical vagotomy (BCV) is
a standard method of inducing ventilatory distress which leads to HMD [J Exp.Med. 66:397, 1937;
Biol.Neonat. 6:340, 1964; Biol. Neonat. 1 1 :6 I. 19671. The lungs of post-vagotomy newborn rabbits
show the lesions of HMD in extent directly proportionate to the percentage of oxygen in polybaric
(0.2 - 3.0 Atm.Abs) mixtures with nitrogen. Avery [Pediatrics 32:801, 19631 found that lesions of
HMD did not form at very low levels of oxygen (3-4% in nitrogen] in various newborn animals.
suggesting that inhalant hypoxia was not a pathogenetic factorper se. The observation of lung injury
proportionate to oxygen percentage indicates the physiological axiom of gas effects by their partial
pressure is an artefact of sea level gas dynamics. The toxic effect of oxygen can be viewed as
nitrogen lack. Some lung injury does occur when only 3 and 7 per cent oxygen in nitrogen is used,
suggesting rather a specific oxygen effect. When nitrogen is replaced by hydrogen, helium. neon,
argon, or sulfur hexafluoride, the extent of lesions often increases, indicating again a fundamental
oxygen-nitrogen interaction. Low level studies with hydrogen and argon are especially instructive
with and without BCV: (1) extremely long survival without BCV in oxygen-argon at 3% and 7 %;
(2) significant but less enhancement of survival in 3% oxygen-hydrogen; (3) no distinction in
survival after BCV for 3% oxygen in nitrogen or hydrogen; (4) a pattern of lesion formation in the
alternative gas mixtures which suggests nitrogen has a partially protective effect along with its
stochastic competition for a conlmon oxygen-nitrogen receptor or transmembrane port; and (5)
generally, the mammalian lung is well adapted by evolution to current atmospheric composition but
at the price of more inhaled oxygen than is required for cellular function [Perspect.Biol.Med. 13:80,
19691, allowing for toxic effects. The distinctions amongst these gases in the biologic sense are due
to differences in their mass, moiloatomic or diatomic structure: possibly viscosity in air passageway
flow, inherent energy state, and at low levcls, in the electron saturation of the outer atomic shell.
Unbuffered oxygen enrichment of air for ventilatory support is fundamentally injurious; hydrogen
has obvious risks in a clinical setting but argon, which is abundant, non-flammable, and relatively
non-toxic, may be the diluent gas of choice for ventilatory support.
Shanklin, D. Radford
Pulmonary fibrosis implies antecedent lung injury which may or may not include inflammatory
responses of the ordinary sort. The onset of breathing at mammalian birth is a different kind of lung
injury, one occasioned by great physical stretch of the collapsed but moist fetal lung, and immediate
exposure to over ten times the level of oxygen resident in the fetal organ. Access to a large archive,
the perinatal mortality review from the Chicago Lying-In Hospital, has provided information very
relevant to these questions, including the first regular documentation of the pulmonary lesion
complex as related to clinical care: beginning in the late 1930s. The lesion complex is called hyaline
membrane disease (HMD) from the condensation at the tissue:gas interface of protein exuded from
the lung and its circulation.
Shanklin, D. Radford
The intracellular dominance of magnesium ion makes clinical assessment difficult despite the critical role of Mg++ in many key functions of cells and enzymes. There is general consensus that serum Mg++ levels are not representative of the growing number of conditions for which magnesium is known to be important. There is no consensus method or sample source for testing for clinical purposes. High intracellular Mg++ in vertebrate embryos results in part from interactions of cations which influence cell membrane transport systems. These are functionally competent from the earliest stages, at least transiently held over from the unfertilized ovum. Kinetic studies with radiotracer cations, osmolar variations, media lacking one or more of the four biological cations, Na+, Mg++, K+, and Ca++, and metabolic poison 0.05 mEq/L NaF, demonstrated: (1) all four cations influence the behavior of the others, and (2) energy is required for uptake and efflux on different time scales, some against gradient. Na+ uptake is energy dependent against an efflux gradient. The rate of K+ loss is equal with or without fluoride, suggesting a lack of an energy requirement at these stages. Ca++ efflux took twice as long in the presence of fluoride, likely due in part to intracellular binding. Mg++ is anomalous in that early teleost vertebrate embryos have an intracellular content exceeding the surrounding sea water, an isolated unaffected yolk compartment, and a clear requirement for energy for both uptake and efflux. The physiological, pathological, and therapeutic roles of magnesium are poorly understood. This will change: (1) when 28Mg is once again generally available at a reasonable cost for both basic research and clinical assessment, and (2) when serum or plasma levels are determined simultaneously with intracellular values, preferably as part of complete four cation profiles. Atomic absorption spectrophotometry, energy-dispersive x-ray analysis, and inductively coupled plasma emission spectroscopy on sublingual mucosal and peripheral blood samples are potential methods of value for coordinated assessments.